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The existence of bladder cancer stem cells (BCSC) has been suggested to underlie bladder tumor initiation and recurrence. Sonic Hedgehog (SHH) signaling has been implicated in promoting cancer stem cell (CSC) self-renewal and is activated in bladder cancer, but its impact on BCSC maintenance is unclear. In this study, we generated a mAb (BCMab1) against CD44+ human bladder cancer cells that recognizes aberrantly glycosylated integrin α3β1. The combination of BCMab1 with an anti-CD44 antibody identified a BCMab1+CD44+ cell subpopulation as BCSCs with stem cell–like properties. Gene expression analysis revealed that the hedgehog pathway was activated in the BCMab1+CD44+ subpopulation and was required for BCSC self-renewal. Furthermore, the glycotransferase GALNT1 was highly expressed in BCMab1+CD44+ cells and correlated with clinicopathologic features of bladder cancers. Mechanistically, GALNT1 mediated O-linked glycosylation of SHH to promote its activation, which was essential for the self-renewal maintenance of BCSCs and bladder tumorigenesis. Finally, intravesical instillation of GALNT1 siRNA and the SHH inhibitor cyclopamine exerted potent antitumor activity against bladder tumor growth. Taken together, our findings identify a BCSC subpopulation in human bladder tumors that appears to be responsive to the inhibition of GALNT1 and SHH signaling, and thus highlight a potential strategy for preventing the rapid recurrence typical in patients with bladder cancer. Cancer Res; 76(5); 1273–83. ©2015 AACR.

Although Hedgehog signaling plays a major role in GLI1 transcription, there is now evidence suggesting that other pathways/genes, such as c-MYC, may also regulate GLI1 expression. We initiated studies in Burkitt lymphoma cells, which constitutively express c-MYC due to a chromosomal translocation, to determine whether Hedgehog or c-MYC regulates GLI1 expression. We show that all Burkitt lymphoma cell lines tested express GLI1, PTCH1, and SMO and that five of six Burkitt lymphomas express GLI1. Exposure to Sonic or Indian Hedgehog or cyclopamine (SMO inhibitor) does not modulate GLI1 expression, cell proliferation, or apoptosis in most Burkitt lymphoma cell lines. Sequence analysis of PTCH1, SMO, and SuFu failed to show mutations that might explain the lack of Hedgehog responsiveness, and we did not detect primary cilia, which may contribute to it. We show that c-MYC interacts with the 5′-regulatory region of GLI1, using chromatin immunoprecipitation (ChIP) assay, and E-box–dependent transcriptional activation of GLI1 by c-MYC in NIH3T3 and HeLa cells. The c-MYC small-molecule inhibitor 10058-F4 downregulates GLI1 mRNA and protein and reduces the viability of Burkitt lymphoma cells. Inhibition of GLI1 by GANT61 increases apoptosis and reduces viability of some Burkitt lymphoma cells. Collectively, our data provide evidence that c-MYC directly regulates GLI1 and support an antiapoptotic role for GLI1 in Burkitt lymphoma. Burkitt lymphoma cells do not seem to be Hedgehog responsive. These findings suggest a mechanism for resistance to SMO inhibitors and have implications for using SMO inhibitors to treat human cancers. Mol Cancer Res; 11(6); 604–15. ©2013 AACR.

Introduction: Epithelial-stromal Hedghehog (HH) pathway signaling, originating from Sonic Hedgehog (SHH) ligand secreted by tumor cells, promotes breast cancer growth and metastasis. Furthermore, high tumor SHH expression is associated with poor prognosis in invasive ductal carcinoma. Studies have also shown high expression of Patched (PTCH) mRNA in SHH expressing tumors as well as cancer stem-like cells. This is in part due to a feedback loop that results in increased PTCH gene expression upon HH signaling. We sought to exploit this feedback loop by using radiolabeled derivative of SHH to identify tumors that have a high level of hedgehog signaling. These agents can potentially be used for molecular imaging of HH induced changes in PTCH expression originating from tumor expressed SHH. Methods: Recombinant N-terminal SHH derivatives were radioiodinated with iodine-131 using the idogen method. Iodinated proteins were obtained with radiolabeling yields of 50–60 % and specific activity of 9.3E10Ci/mol. Receptor binding studies were performed on breast cancer cell lines with varying SHH expression. Studies were also conducted on cells cultured in stem cell promoting conditions (mammospheres), after exposure to ionizing radiation or treatment with the hedgehog inhibitor cyclopamine. Scatchard plots were generated using Graph pad software. Pilot studies were conducted with 131I-SHH to test the potential of imaging PTCH receptor expression in a rat model of breast cancer. Fisher rats bearing breast cancer xenografts were injected with approximately 250 microCi of 131I-SHH. Planar scinitigraphy was conducted at 2, 4 and 24 hours. Studies were performed with iodinated BSA as a control for extracellular perfusion and retention. Tissue biodistribution studies were also performed using this model. Results: Receptor binding of 131I-SHH was significantly increased in cell lines with high endogenous HH pathway activation. Receptor binding was also increased after exposure to ionizing radiation and significantly decreased following treatment with hedgehog inhibitors. Scatchard analysis revealed up to a 14 fold increase in PTCH receptor sites in mammospheres compared to the entire breast cancer population. A 10-fold increase in PTCH receptor binding sites was also observed in cells after exposure to ionizing radiation. In vivo imaging and biodistribution studies revealed significant accumulation of 131I-SHH within tumor tissue as compared to normal organs. Tumor:muscle ratios were approximately 8:1 at 4 hours, while tumor to blood and tumor to bone were 2:1 and 5:1 respectively. Significant uptake was also observed in liver tissue, as a result of protein degradation and excretion and endogenous PTCH expression. Conclusions: These studies show that expression of the PTCH receptor is increased in cells with active HH signaling and in breast cancer mammospheres. Our findings also show that PTCH receptor expression is decreased upon treatment with HH signaling inhibitors. Preliminary imaging studies show that 131I-SHH is capable of in vivo detection of breast tumors with high hedgehog signaling. Our data suggests that radiolabeled SHH derivatives may provide a method to follow epithelial-stromal HH interactions and determine response to SHH targeted therapies. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-09-05.

Activation of the Hedgehog (Hh)-signaling pathway due to deficiency in the Hh receptor Patched1 (Ptch) is the pivotal defect leading to formation of basal cell carcinoma (BCC). Recent reports provided evidence of Ptch-dependent secretion of vitamin D3-related compound, which functions as an endogenous inhibitor of Hh signaling by repressing the activity of the signal transduction partner of Ptch, Smoothened (Smo). This suggests that Ptch-deficient tumor cells are devoid of this substance, which in turn results in activation of Hh-signaling. Here, we show that the application of the physiologically active form of vitamin D3, calcitriol, inhibits proliferation and growth of BCC of Ptch mutant mice in vitro and in vivo. This is accompanied by the activation of the vitamin D receptor (Vdr) and induction of BCC differentiation. In addition, calcitriol inhibits Hh signaling at the level of Smo in a Vdr-independent manner. The concomitant antiproliferative effects on BCC growth are stronger than those of the Hh-specific inhibitor cyclopamine, even though the latter more efficiently inhibits Hh signaling. Taken together, we show that exogenous supply of calcitriol controls the activity of 2 independent pathways, Hh and Vdr signaling, which are relevant to tumorigenesis and tumor treatment. These data suggest that calcitriol could be a therapeutic option in the treatment of BCC, the most common tumor in humans. Mol Cancer Ther; 10(11); 2179–88. ©2011 AACR.

Canonical Hedgehog (HH) signaling is characterized by Smoothened (Smo)-dependent activation of the transcription factors Gli1 and Gli2, which regulate HH target genes. In human colon carcinoma cells, treatment with the Gli small-molecule inhibitor GANT61 induces extensive cell death in contrast to the Smo inhibitor cyclopamine. Here we elucidate cellular events upstream of cell death elicited by GANT61, which reveal the basis for its unique cytotoxic activity in colon carcinoma cells. Unlike cyclopamine, GANT61 induced transient cellular accumulation at G1–S (24 hours) and in early S-phase (32 hours), with elevated p21Cip1, cyclin E, and cyclin A in HT29 cells. GANT61 induced DNA damage within 24 hours, with the appearance of p-ATM and p-Chk2. Pharmacologic inhibition of Gli1 and Gli2 by GANT61 or genetic inhibition by transient transfection of the Gli3 repressor (Gli3R) downregulated Gli1 and Gli2 expression and induced γH2AX, PARP cleavage, caspase-3 activation, and cell death. GANT61 induced γH2AX nuclear foci, while transient transfection of Gli3R showed expression of Gli3R and γH2AX foci within the same nuclei in HT29, SW480, and HCT116. GANT61 specifically targeted Gli1 and Gli2 substantiated by specific inhibition of (i) direct binding of Gli1 and Gli2 to the promoters of target genes HIP1 and BCL-2, (ii) Gli-luciferase activity, and (iii) transcriptional activation of BCL-2. Taken together, these findings establish that inhibition of HH signaling at the level of the GLI genes downstream of Smo is critical in the induction of DNA damage in early S-phase, leading to cell death in human colon carcinoma cells. Cancer Res; 71(17); 5904–14. ©2011 AACR.

Hedgehog (Hh) signaling plays an important role in prostate development and is also a characteristic feature of prostate cancer (PCa). The ability of Hh pathway activation to promote cell invasiveness, epithelial to mesenchymal transition and metastasis suggest that targeting Hh pathway may provide an important clinical avenue for the treatment of advanced PCa. Loss of PTEN function is considered a characteristic feature of advanced PCa. To gain insight into the status of Hh signaling in PCa in relation to PTEN status we selected three cell lines based on their PTEN expression. Gli1 mRNA was upregulated 2000 fold in PC3 (PTEN-/- null) cells, 800 fold in DU145 (PTEN+/-) cells when compared with normal prostate epithelial cells suggesting that PTEN loss is associated with increased Hh signaling. We also observed significant increase in the mRNA levels of Hh ligand (Shh), Hh receptors (PTCH2 and SMO) and effectors of Hh signaling (Gli1 and Gli2) in the prostate tissues of PTEN knockout mice. A 400 fold increase in the mRNA levels of Gli1 and 15 fold increase in the levels of Gli2 mRNA levels was also observed. We also observed significant decrease in the mRNA levels of the negative regulators of Hh signaling, HHIP and SuFu in the prostate tissues of PTEN knockout mice with similar inhibition in the transcript levels of GAS1. We next investigated the effect on Hh signaling in PCa cells treated with itraconazole and cyclopamine alone and in combination. PCa 22Rν1 and PC-3 cells transfected with Gli-dependent luciferase reporter construct were treated with itraconazole (1 and 2 μM), cyclopamine (5 and 10 nM) and combination of itraconazole (1 μM) and cyclopamine (5 nM) for 40 hours. Gli-dependent luciferase reporter activity was inhibited by itraconazole and cyclopamine treatments; however the inhibition was significantly greater in combination. Treatment of PCa cells with the combination of itraconazole and cyclopamine led to increased cell growth inhibition that was greater than the sum of the individual agents alone suggesting synergism. We next analyzed the effect of the combination on the growth of CWR22Rν1 tumors under in vivo conditions using athymic nude mice. In control animals, targeted tumor volume of 1200 mm3 was reached at 6 weeks post cell inoculation and significantly later time in animals treated with itraconazole alone (9 weeks) and cyclopamine alone (8 weeks). Notably, tumor volumes stayed under 165 mm3 in mice that received both itraconazole and cyclopamine. In the prostate specific Pten knockout mice combination of itraconazole and cyclopamine produced 57% inhibition of tumor growth compared to 30% in cyclopamine alone and 45% in itraconazole alone treated mice. These data show that a combination of itraconazole and cyclopamine is considerably more efficient in inhibiting tumor growth highlighting the synergistic efficacy of this combination for the treatment of PCa. Citation Format: Vaqar M. Adhami, Imtiaz A. Siddiqui, Mohammad Imran Khan, Islam Rady, Leanna Sako, Hasan Mukhtar. Hedgehog pathway inhibitors itraconazole and cyclopamine produce synergistic suppression of Pten deficient prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5808.

Sonic hedgehog signaling pathway is an important regulator during the initiation and progression of pancreatic cancer, and sustained activation of this pathway contributes to excessive deposition of tumor stroma and maintenance of tumor-initiating cells. Cyclopamine (CPA) is a potent inhibitor of the SHH pathway. In this work, we encapsulated both paclitaxel (PTX) and CPA in a long-circulating polymeric micelles to obtain M-CPA/PTX. In a genetically engineered mouse model of pancreatic ductal adenocarcinoma (PDAC) (KPC model), systemic administration of M-CPA/PTX significantly prolonged the median survival of mice with well-established tumors from 23 days with nab-paclitaxel or gemcitabine to 58 days post-enrollment (p Citation Format: Jun Zhao, Zhilan Xiao, Willem Overwijk, Chun Li. Pharmacological modulation of PDAC stroma to enhance checkpoint blockade immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1014.

Many botanical compounds have been proposed to prevent cancer. We investigated the cancer treatment and prevention abilities of apigenin, baicalein, curcumin, epigallocatechin 3-gallate (EGCG), genistein, quercetin, and resveratrol both in vivo in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice as well as in vitro in prostate cancer cell lines. In our experiments, these seven compounds act similarly to the Hedgehog antagonist cyclopamine, a teratogenic plant alkaloid, which had been previously shown to “cure” prostate cancer in a mouse xenograft model. With IC50 values ranging from

The present study has been undertaken to establish the therapeutic benefit of cotargeting epidermal growth factor receptor (EGFR) and sonic hedgehog pathways by using gefitinib and cyclopamine, respectively, for improving the efficacy of the current chemotherapeutic drug docetaxel to counteract the prostate cancer progression from locally invasive to metastatic and recurrent disease stages. The data from immuofluorescence analyses revealed that EGFR/Tyr1173-pEGFR, sonic hedgehog ligand, smoothened coreceptor, and GLI-1 were colocalized with the CD133+ stem cell–like marker in a small subpopulation of prostate cancer cells. These signaling molecules were also present in the bulk tumor mass of CD133− prostate cancer cells with a luminal phenotype detected in patient's adenocarcinoma tissues. Importantly, the results revealed that the CD133+/CD44high/AR−/low side population (SP) cell fraction endowed with a high self-renewal potential isolated from tumorigenic and invasive WPE1-NB26 cells by the Hoechst dye technique was insensitive to the current chemotherapeutic drug, docetaxel. In contrast, the docetaxel treatment induced significant antiproliferative and apoptotic effects on the CD133−/CD44low/AR+ non-SP cell fraction isolated from the WPE1-NB26 cell line. Of therapeutic interest, the results have also indicated that combined docetaxel, gefitinib, and cyclopamine induced greater antiproliferative and apoptotic effects on SP and non-SP cell fractions isolated from WPE1-NB26 cells than individual drugs or two-drug combinations. Altogether, these observations suggest that EGFR and sonic hedgehog cascades may represent the potential therapeutic targets of great clinical interest to eradicate the total prostate cancer cell mass and improve the current docetaxel-based therapies against locally advanced and invasive prostate cancers, and thereby prevent metastases and disease relapse. Mol Cancer Ther; 9(3); 617–30

There is an enormous gap between the antiproliferative and in vivo antitumor efficacy of gemcitabine in cell line-based models and its clinical efficacy. This may be due to insensitiveness of the precursor, cancer stem cell (CSC) compartment to cytotoxic agents. The hedgehog pathway is associated with CSC signaling and control. We used a direct xenograft model of pancreatic cancer and a two-stage approach was used to test the hypotheses that targeting CSC could increase the efficacy of gemcitabine. Tumors from a gemcitabine-sensitive xenograft were treated with gemcitabine first, and randomized, after tumor regression to continuing treatment with gemcitabine, a hedgehog inhibitor alone or in combination with gemcitabine. We tested markers described as associated with CSC such as CD24, CD44, ALDH, nestin, and the hedgehog pathway. After induction with gemcitabine, treated tumor showed an enrichment in CSC markers such as ALDH and CD24. Subsequently, a release from gemcitabine prompted a repopulation of proliferating cells and a decrease in such markers to equilibrate from pretreatment levels. Combined treatment with gemcitabine and cyclopamine induced tumor regression and decrease in CSC markers and hedgehog signaling. Cytoplasmic CD24 and ALDH were inversely and strongly associated with growth and were expressed in a minority of cells that we propose constitute the CSC compartment. Hedgehog inhibitors as part of a dual compartment therapeutic approach were able to further reduce tumor growth and decreased both static and dynamic markers of CSC. Direct tumor xenografts are a valid platform to test multicompartment therapeutic approaches in pancreatic cancer. [Mol Cancer Ther 2009;8(2):310–4]

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by an accumulation of neoplastic B cells due to their resistance to apoptosis and increased survival. Among various factors, the tumor microenvironment is known to play a role in the regulation of cell proliferation and survival of many cancers. However, it remains unclear how the tumor microenvironment contributes to the increased survival of B-CLL cells. Therefore, we studied the influence of bone marrow stromal cell–induced hedgehog (Hh) signaling on the survival of B-CLL cells. Our results show that a Hh signaling inhibitor, cyclopamine, inhibits bone marrow stromal cell–induced survival of B-CLL cells, suggesting a role for Hh signaling in the survival of B-CLL cells. Furthermore, gene expression profiling of primary B-CLL cells (n = 48) indicates that the expression of Hh signaling molecules, such as GLI1, GLI2, SUFU, and BCL2, is significantly increased and correlates with disease progression of B-CLL patients with clinical outcome. In addition, SUFU and GLI1 transcripts, as determined by real-time PCR, are significantly overexpressed and correlate with adverse indicators of clinical outcome in B-CLL patients, such as cytogenetics or CD38 expression. Furthermore, selective down-regulation of GLI1 by antisense oligodeoxynucleotides (GLI1-ASO) results in decreased BCL2 expression and cell survival, suggesting that GLI1 may regulate BCL2 and, thereby, modulate cell survival in B-CLL. In addition, there was significantly increased apoptosis of B-CLL cells when cultured in the presence of GLI1-ASO and fludarabine. Together, these results reveal that Hh signaling is important in the pathogenesis of B-CLL and, hence, may be a potential therapeutic target. (Mol Cancer Res 2008;6(12):1928–36)

Purpose: In humans, several distinctive cancers result from mutations that aberrantly activate hedgehog (HH) signal transduction. Here, we investigate the role of HH signaling in ovarian cancer. Experimental Design: We assessed the expression of different components of hedghehog pathway in primary tumor samples and cell lines. We used specific hedghehog pathway blocker to study the effect on clonal growth and proliferation of ovarian cancer cell both in vitro and in vivo. Results: We show that the up-regulation of several HH pathway components is a common feature of primary ovarian tumors and cell lines. However, expression of PATCHED1 (PTCH1), a direct transcriptional target of the HH pathway, is down-regulated in ovarian cancer in direct contrast to the expression observed in other adult solid tumors. Cyclopamine, a specific HH pathway inhibitor, inhibits the proliferation and clonal growth of ovarian tumor cells in vitro and arrests ovarian tumor growth in vivo. Expression of BMI-1, a polycomb gene, is down-regulated in ovarian cancer cells following cyclopamine treatment. Overexpression of PTCH1 phenocopied the effects of cyclopamine; it down-regulated BMI-1 and reduced clonal growth in ovarian cancer cell lines. Furthermore, knocking down BMI-1 using small interfering RNA also inhibited the clonal growth of all the ovarian cancer cell lines tested. Conclusions: In brief, the constitutive low-level expression of PTCH1 contributes to proliferation and clonal growth of ovarian cancer cells by an aberrant HH signal. Because the HH pathway can be inhibited by specific inhibitors, these findings point toward possible new treatments to inhibit ovarian cancer growth.

The sonic hedgehog (Shh) pathway contributes to the initiation and progression of tumors with various origins when aberrantly activated. In this study, we investigated if the Shh pathway is important for the proliferation of hepatocellular carcinoma (HCC) cells and also began to identify which components of the pathway play a pivotal role in the biology of HCC. Expression levels of components in the pathway were measured, and glioma-associated oncogene (Gli) 2 levels were found to be considerably higher in human HCC lines compared with normal liver. Gli2 levels were also higher in tumor tissue from HCC patients compared with normal liver. Antisense oligonucleotides (ASO) were used to specifically down-regulate Gli2, and this led to decreased proliferation of various HCC cell lines. However, inhibition of Gli1 and Gli3 with ASOs did not decrease proliferation in most HCC cell lines and inhibitors targeting the upstream components of the pathway, including smoothened (Smo), displayed antiproliferative effects in only a subset of HCC cell lines. Moreover, in cancer cells harboring Smo mutations or unresponsive to the Smo inhibitor 3-keto-N-aminoethylaminoethylcaproyldihydrocinnamoyl cyclopamine, the Gli2 ASO was still able to inhibit proliferation. The importance of Gli2 in HCC proliferation was further confirmed by the changes in expression levels of genes, such as Bcl-2, c-Myc, and p27, following suppression of Gli2 expression. Taken together, these results suggest that, among the Gli transcription factors, Gli2 plays a predominant role in the proliferation of HCC cells and the suppression of Gli2 expression may provide a useful therapeutic option for the treatment of HCC. [Cancer Res 2007;67(8):3583–93]

The epidermal growth factor receptor (EGFR) and hedgehog cascades provide a critical role in prostate cancer progression and contribute to the resistance to clinical therapies and disease relapse. Therefore, we evaluated, for the first time, the antiproliferative and cytotoxic effects induced by a combination of selective inhibitors of EGFR tyrosine kinase and smoothened hedgehog signaling element, gefitinib and cyclopamine, with a current chemotherapeutic drug used in the clinics, docetaxel, on some metastatic prostate cancer cell lines. Immunohistochemical analyses revealed that sonic hedgehog (SHH) expression was enhanced in 39% of primary prostatic adenocarcinomas (Gleason scores 4–10) compared with the corresponding normal tissues of the same prostate gland from 32 prostate cancer patients. The confocal microscopy and Western blot analyses have also indicated the high expression levels of SHH and EGFR in metastatic LNCaP, DU145, and PC3 cells. Moreover, the results revealed that the drugs, alone or in combination, at lower concentrations inhibited the growth of EGF plus SHH–stimulated and serum-stimulated androgen-responsive LNCaP-C33 and androgen-independent LNCaP-C81, DU145, and PC3 cells. Importantly, the combined docetaxel, gefitinib, and cyclopamine also caused a higher rate of apoptotic death of prostate cancer cells compared with individual agents. The cytotoxic effects induced by these drugs in PC3 cells seem to be mediated in part through the cellular ceramide production and activation of caspase cascades via a mitochondrial pathway and the release of cytochrome c into the cytosol. Additionally, the combined agents were more effective at suppressing the invasiveness of PC3 cells through Matrigel in vitro than the single drugs. These findings indicate that the combined use of inhibitors of EGF-EGFR and hedgehog signaling with docetaxel could represent a more promising strategy for treatment in patients with metastatic and androgen-independent prostate cancer. [Mol Cancer Ther 2007;6(3):967–78]

The existence of Cancer Stem Cells (CSCs) has been proven extensively in recent years. CSCs have been positively identified in, and isolated from colon cancer. They have been found to be resilient to therapy and are highly metastatic and it has been suggested that this could be attributed to the presence of CSCs expressing cell surface CD133. An active Hedgehog-Gli pathway has been detected in human colon carcinoma cells as well as their stem cells. Sonic Hedgehog (Shh) inhibitors such as cyclopamine have been successful in the treatment of other cancer types. CSCs were isolated from HT29 and DLD1 cells using the CD133 surface protein and these cells were further characterized via their expression of key stem cell markers using fluorescence microscopy. These markers included c-Myc, KLF4 and alkaline phosphatase, all of which showed elevated expression levels in the CSC fraction. The growth pattern of CSCs in comparison to non-CSCs was analyzed using cell-impedance (xCELLigence) assays, with clear differences noted in their growth rates. Adhesion assays revealed that although CSCs have a lower proliferation rate, their adhesive potential proved to be greater, possibly due to them being highly metastatic. The effects of the Shh inhibitors cyclopamine and SANT-2 on cellular adhesion of colon CSCs were compared to the Shh pathway control molecule tomatidine, and a marked decrease in adhesion potential was noted. With regard to cell invasion, this was significantly decreased when CSCs were treated with the small molecule inhibitors. In cell migration/scratch assays a similar trend was seen, wherein cell migration was retarded by Hh pathway inhibition. Using confocal microscopy, the cell surface expression of Shh and CD133 was observed following treatment with cyclopamine and while found to significantly reduce Shh expression, CD133 expression remained unaffected. Considering these in vitro results small molecule inhibitors targeting the Shh pathway appear to be promising therapeutic tools for the treatment of metastatic colon CSCs. Citation Format: Aadilah Omar, Clement Penny, Paul Ruff. Inhibition of Sonic Hedgehog Pathway by small molecule inhibitors: Targeting colon cancer stem cells [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr A21.

[Background] Hedgehog (Hh) signaling is activated under hypoxic condition in cancerous tissue. This is thought to be one of the mechanisms of the induction of malignant phenotype of cancers. We have shown that Hh inhibitor decreases proliferation, invasiveness, and tumorigenesis. We have also revealed that Hh signaling plays pivotal roles for the maintenance of function in activated lymphocytes and dendritic cells under hypoxia. Recently, immune checkpoint inhibitor takes much attention, however, response rate is still limited. The mechanisms regulating PDL-1 expression also remains unclear. In this study, to increase reponse rate of immune checkpoint inhibitor, we investigated whether hypoxia-induced activation of Hh signaling contributes to PDL-1 expression in cancer and whether it affects the antitumor function of activated lymphocytes. [Materials and Methods] Pancreatic cancer cell line, Panc-1, gallbladder cancer cell line, NOZ and GBd, and lung small cell cancer cell line, SBC-5 were used as target cancer cells. Human lymphocytes derived from PBMC were activated by using anti CD3 mAb and IL-2, and were used as effector cells. For normoxic conditions, cells were cultured in 5% CO2 and 95% air. For hypoxic conditions, cells were cultured in 1% O2, 5% CO2, and 94% N2 using a multigas incubator. For Hh signaling inhibition, cyclopamine, polysaccharide-K (PSK), and small interfering RNA targeting Gli1, Smo, MAML3, and RBPJ were used. Cell numbers were counted by light microscope. Expression of cell surface molecules was estimated by FACS. [Results] 1) Hypoxia augmented PDL-1 expression in all 4 cancer cell lines. 2) Inhibition of Hh signaling using MAML3 siRNA, cyclopamine and PSK reduced PDL-1 expression under hypoxia in all 4 cancer cell lines. 3) When activated lymphocytes were cocultured with cancers treated with a Hh inhibitor, activated lymphocyte cell numbers significantly increased under hypoxia. 4) In contrast, this increase was abrogated when cancer cells were treated with a PDL-1 neutralizing antibody. 5) When activated lymphocytes were cocultured with cancers treated with a Hh inhibitor and/or anti-PDL-1 Ab, the percent of CD8+ lymphocytes decreased in both the cyclopamine- and anti-PDL-1-treated groups under hypoxic conditions, while there was no significant change in CD3 expression. 6) NKG2D expression increased on activated lymphocytes in anti PDL-1Ab-treated group. [Conclusion] These results suggest that Hh signaling is one of regulatory pathways of PDL-1 expression under hypoxia and that inhibiting Hh signaling may induce lymphocyte antitumor activity. Therefore, Hh inhibition could be a promising drug target, not only as a direct tumor suppressor agent, but also as a PDL-1 inhibitor. Citation Format: Hideya Onishi, Akiko Fujimura, Yasuhiro Oyama, Makoto Kawamoto, Akio Yamasaki, Takashi Morisaki. Hedgehog signaling augments PDL-1 expression in cancer cells under hypoxic condition to inhibit antitumor effects by activated lymphocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 589. doi:10.1158/1538-7445.AM2017-589

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has been known as one of the most effective cancer treatment owing to its selective killing effect of cancer but not normal cells. However it has been reported that several gastric cancer cells showed resistance to TRAIL because of scarcity of death receptor 5 (DR5) expressed on the cell surface. In this study, we elucidated cyclopamine sensitizes to TRAIL-induced apoptosis by elevating the expression level of DR5 in gastric cancer cells. Interestingly, survivin hampers existence of DR5 protein under normal condition. Cyclopamine decreases expression level of survivin. It is a reason for cyclopamine to act as a TRAIL sensitizer. Mechanistically, cyclopamine induces endoplasmic reticulum (ER) stress by Reactive Oxygen species (ROS), and CHOP, which as a last protein of ER stress pathway regulates stability of survivin protein by proteasome degradation. Taken together, our results indicate that cyclopamine can be used for the purpose of combination therapy in TRAIL-resistant gastric cancer cells. Citation Format: Yoo Jin Na, Dae-Hee Lee, Jung Lim Kim, Bo Ram Kim, Seong Hye Park, Min Jee Jo, Yoon A Jeong, Sang Cheul Oh. Cyclopamine sensitizes endoplasmic reticulum stress-mediated increase of death receptor 5 and survivin degradation in trail-resistant gastric cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2318. doi:10.1158/1538-7445.AM2017-2318

We have performed a genome-wide screen for pathways that are synthetic lethal with TP53 mutations in colon cancer. RKO colon cancer cells are wild type for TP53, and display robust p53-dependent growth arrest and apoptosis. We utilized an isogenic pair of RKO cells differing only in TP53 gene status (wild type vs homozygous null derivative) to screen a genome-wide GeCKO CRISPR library. After infection of these cells with the library and culturing for one week in vitro, we performed deep amplicon sequencing to identify CRISPR guide RNAs that were underrepresented in TP53 knockout cells. Multiple CRISPR guide RNAs targeting the CHEK1 and SHH genes were underrepresented in TP53 knockout cells. To validate these targets, we used CRISPR genome engineering to create an independent panel of six RKO clones with biallelic disruption of TP53, and tested small molecule inhibitors of CHEK1 (UCN01) and the SHH receptor SMO (Cyclopamine). TP53 null cells were tenfold more sensitive to UCN01, and 100-fold more sensitive to Cyclopamine, compared to TP53 wild type cells. Finally, the relevance of these findings to human colon cancers was confirmed by the establishment and treatment of colon cancer organoid avatars with either wild type or mutant TP53. Normal colon organoids, and tumor organoids with wild type TP53 were unaffected by either drug, whereas TP53 mutant organoids were significantly growth inhibited. Future studies will focus on testing these and additional targets, alone and in combination, against primary colon tumor and normal avatars and other preclinical models of TP53-mutant colon cancer. Citation Format: Charles C. Weige, Changlong Liu, Erin L. Anderson, Carolyn E. Banister, Joseph Richardson, Carlo Contreras, Phillip J. Buckhaults. TP53 synthetic lethal targets in colon cancer. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr B14.

We have performed a genome-wide synthetic lethal screen for pathways that are essential for the survival of colon cancer cells with TP53 mutations. RKO colon cancer cells are wild-type for TP53, and display robust p53-dependent growth arrest and apoptosis. We have utilized an isogenic pair of RKO cells differing only in TP53 gene status (Wild Type vs homozygous null derivative) to screen a genome-wide GeCKO CRISPR library and identified pathways that are novel therapeutic targets for TP53-mutant colon cancers. After infection of these cells with the GeCKO CRISPR library and one week of culturing in vitro, we performed deep GeCKO CRIPSR amplicon sequencing to enumerate and identify all CRISPRs that were underrepresented in TP53 mutant compared to wild type cells. We identified multiple CRISPRs targeting the CHEK1 and the SHH genes which were underrepresented in TP53 knockout cells. We then tested small molecule inhibitors of CHEK1 (UCN01) and the SHH receptor SMO (Cyclopamine) against isogenic pairs of RKO, HCT116, and DLD1 cells and confirmed that each molecule is significantly more toxic to cells with mutant TP53 than to those with wild-type TP53. Future studies will focus on testing these and additional targets alone and in combination against primary colon tumor and normal avatars, and preclinical models of TP53-mutant colon cancer. Citation Format: Charles C. Weige, Erin L. Anderson, Joseph Richardson, Carlo Contreras, Phillip J. Buckhaults. Synthetic lethal screen to identify novel therapies targeting TP53-mutant colon cancer cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr B10.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor; it is genetically defined into 4 subgroups which defines the clinical course of the disease. Despite current treatment modalities such as surgical resection, radiation, and chemotherapy, there are still significant morbidities and adverse side-effects. Recently defined genetic pathways could possibly offer a better management and treatment of patients with MB. The HIPPO signaling pathway has recently been shown to intersect with PI3K and Sonic Hedgehog (SHH) pathways to modulate mechanistic target of rapamycin (mTOR), an important regulator in cell growth and migration. The aim of this study is to elucidate the interaction of Hippo and Akt/mTOR signaling pathways with SHH pathways in MB, and define the potential downstream targets of therapy by using combined inhibitors. In order to achieve our goals, we evaluated the expression of activated AKT/mTOR in MB tumors using immunohistochemistry (IHC), along with the expression of transcriptional co-activators yes-associated protein (YAP), a down-stream effector of the HIPPO pathway. Scratch and chemotactic migration were used to assess the motility of MB cells following treatment with inhibitors of the aforementioned pathways. Cell cycle analysis and proliferation were measured using EdU and MTT techniques, respectively. YAP-SiRNA treatment was used to inhibit HIPPO pathway. MicroRNA-29, which mediates the mTOR pathway by modulating PTEN levels, was investigated. Results demonstrated: 1. A significant number of MBs expressed activated pAKTSer473 and pmTORSer2448, along with YAP, the downstream effector of the HIPPO pathway; 2. Treatment with inhibitors of PI3K/AKT (LY294002) and mTOR (rapamycin), given with EGF, significantly reduced cellular motility; 3. SHH inhibitor cyclopamine given with LY294002 or rapamycin significantly reduced cellular motility; 4. Cell proliferation was suppressed by inhibition of the HIPPO pathway using 3 unique 27mer duplexes of YAP1 siRNA treatments; 5. Combined treatment of cyclopamine with either LY294002 or rapamycin caused maximum suppression of cellular growth. In conclusion, these results demonstrate that the Hippo and PI3K/Akt/mTOR pathways are over-expressed in MB, and that the combined targeting of mTOR/HIPPO pathway with SHH inhibitor may provide alternative strategies for the treatment of MB, and offers a targeted therapy for improved prognosis. Citation Format: Jennifer S. Ronecker, Paul Lee, Sudeepta Sridhara, Michael LaBagnara, Raj Murali, Meena Jhanwar-Uniyal. The intersection of the PI3K/mTOR and HIPPO pathways: a potential therapeutic target for medulloblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1280.

The Hedgehog (Hh) pathway is a major regulator of many fundamental processes in mammalian embryonic development. These processes include stem cell maintenance, determination of cell fate, tissue polarity, cell survival, cell differentiation and proliferation. Although this pathway is usually silenced in adult tissues, constitutive activation of the Hh pathway can lead to tumorigenesis. This aberrant characteristic can be observed in different types of human cancer including basal cell carcinomas, medulloblastoma, lung cancer, ovarian cancer, gastrointestinal cancer, breast and prostate cancer. This pathway has been shown to regulate proliferation of cancer stem cells and increase tumor invasiveness. Targeted inhibition of Hh signaling pathway is shown to be effective in cancer treatment and prevention. Therefore, the discovery of Hh pathway inhibitors can pave the way of finding effective cancer treatments. The steroidal alkaloid cyclopamine was the first compound found to inhibit Hh signaling and has been invaluable for understanding the function of Hh signaling in development and cancer. Cyclopamine tartrate (CycT) is an improved analogue of cyclopamine with increased water solubility and bioavailabilty. We performed numerous experiments to examine the effect of CycT in inhibiting Hh signaling on an array of non-small-cell lung cancer (NSCLC) lines. We discovered that CycT has a novel activity in disrupting mitochondrial function and aerobic respiration. Our data show that CycT causes a substantial decrease in aerobic respiration of NSCLC cells, similar to the effect of glutamine deprivation. Mitotracker staining showed that CycT causes mitochondrial fragmentation by increasing mitochondrial fission, which is indicated by the localization of DRP1 at fission sites. Further experiments showed that CycT increases ROS generation, which is presumably a consequence of mitochondrial hyperpolarization. Concomitantly, CycT causes apoptosis and suppress proliferation of NSCLC cells. Citation Format: Md Maksudul Alam, Sagar Sohoni, Sarada Preeta Kalainayakan, Massoud Garrossian, Li Zhang. Cyclopamine tartrate, an anti-cancer agent targeting Hedgehog signaling, strongly interferes with mitochondrial function and suppresses aerobic respiration. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3086.

Sonic hedgehog (SHH) signaling pathway is an important regulator during the initiation and progression of pancreatic cancer, and sustained activation of this pathway contributes to excessive deposition of tumor stroma and maintenance of tumor-initiating cells. Cyclopamine, a potent inhibitor of the SHH pathway, has shown promising antitumor efficacy. However, cyclopamine is insoluble in water and is highly toxic, and cyclopamine monotherapy did not effectively inhibit tumor growth in a range of preclinical pancreatic cancer models. We hypothesized that polyion complex polymeric micelles that simultaneously deliver cyclopamine and paclitaxel (M-CPA/PTX) would display greater efficacy against pancreatic tumor than micelles loaded with cyclopamine alone (M-CPA) or paclitaxel alone (M-PTX). We tested this hypothesis in cell cultures and in an orthotopic human pancreatic cancer Miapaca-2 xenograft model and a genetically engineered mouse model of pancreatic ductal carcinoma (PDAC), the KPC model. Our results showed that M-CPA interrupted the SHH pathway by antagonizing the smoothened receptor, reduced expression of Gli-1 transcription factor, and inhibited proliferation of Miapaca-2 cells and stroma-producing human pancreatic stellate cells. In Miapaca-2 cells, M-CPA decreased the fraction of CD133+ cells (cancer stem cells) and decreased tumorsphere formation, whereas M-TXL increased the fraction of CD133+ cells and tumorsphere formation. M-CPA negated the stimulation of human pancreatic stellate cells by exogenous SHH ligands. In vivo, M-CPA/PTX reduced tumor cell proliferation, depleted tumor stroma, and increased tumor vasculature density, leading to effective inhibition of tumor growth in Miapaca-2 xenografts. In the KPC model, M-CPA/PTX significantly prolonged media survival of mice with PDAC compared to non-treatment control mice. In conclusion, M-CPA/PTX was an effective strategy to treat pancreatic cancer. (supported in part by the Viragh Family Foundation and the John S. Dunn Foundation) Citation Format: Jun Zhao, Chunhui Wu, Xiaoxia Wen, Junjie Li, Qian Huang, Ying Ma, Stephen Ullrich, Huamin Wang, Jason Fleming, Chun Li. Pancreatic cancer therapy using polyion micelles co-loaded with cyclopamine and paclitaxel. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1318.

Activation of sonic hedgehog (Shh) signaling has been involved in progression of various cancers. Cyclopamine uses as effective treatment for cancers in which hedgehog signaling is overexpressed. In this study, we elucidated that cyclopamine sensitizes to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in gastric cancer cells. Single treatment with cyclopamine or TRAIL could not induce significant cytotoxicity in gastric cancer cells. However, combination treatment of TRAIL with cyclopamine effectively led to caspase dependent apoptosis. We found that cyclopamine increased ER (Endoplasmic Reticulum) stress level in gastric cancer cells, and using ER stress inhibitor attenuated the cell death by cyclopamine. As further underlying mechanism, induction of ER stress by cyclopamine caused upregulation of JNK (c-Jun N-terminal kinases) protein, and then accumulation of p53 protein. We conducted experiment using p53 wild-type and p53-mutated gastric cancer cells, and this of particular importance since p53 wild-type gastric cancer cells had more significant efficacy than p53-mutated cells by cyclopamine. Accumulation of p53 increased DR5 protein. Taken together, we showed that cyclopamine sensitizes to TRAIL-induced apoptosis in gastric cancer cells. Citation Format: Yoo Jin Na, Dae-Hee Lee, Jung Lim Kim, Bo Ram Kim, Seong Hye Park, Byung-Wook Min, Sun Il Lee, SangHee Kang, Sung Yup Joung, Suk Young Lee, Hong-Jun Kim, Sang Cheul Oh. Cyclopamine enhances TRAIL-induced apoptosis by induction of DR5 via ER stress in gastric cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3512.

Introduction: Aberrant hedgehog (Hh) pathway activation contributes to the pathogenesis of multiple cancers. Currently available Hh pathway inhibitors target Smoothened (Smo) which can acquire mutations causing drug resistance. Therefore, a major challenge is to identify compounds that inhibit Hh signaling downstream of Smoothened. Methods: We conducted a high-throughput screening (HTS) campaign of 337,000 small molecules using a Hh-dependent differentiation assay of C3H10T1/2 cells into osteoclasts. Primary active screening hits were validated by a cell-based assay cascade including motor neuron differentiation from mouse embryonic stem cells, Gli reporter activation in ShhLight2 cells and proliferation of heterozygous Patched (PTCH+/-) medulloblastoma (MB) cells. Smo binding was monitored in membrane-based Cyclopamine competition assays. To identify the underlying target identification of promising hits, affinity chromatography was performed followed by label-free quantitative (LFQ) tandem mass spectrometry. Effects on cytoplasmic dynein were examined in Smo trafficking and in vitro microtubules motility assays as well as by live cell imaging of labeled endosome movement. Results: We identified dynarrestin, a novel aminothiazole that potently blocks Hh signaling and the proliferation of Hh-dependent PTCH+/- MB tumor cells (IC50∼70nM). Dynarrestin does not bind to Smo and is able to suppress the Hh pathway in the presence of the Smo agonist SAG and following the knockdown of the Hh pathway suppressor SUFU indicating that dynarrestin inhibits Hh signaling downstream of both Smo and SUFU. Chemical LFQ proteomics identified cytoplasmic dynein as the target of dynarrestin. Dyneins convert energy from ATP hydrolysis into motor activity and are essential for many cellular processes, including Hh signaling. Unlike other dynein inhibitory molecules, e.g. Ciliobrevins, dynarrestin inhibits Smoothened trafficking but not ciliogenesis. We further demonstrate that dynarrestin reversibly interferes with endosome movement, proper mitotic spindle orientation, and dynein-based microtubule translocation in vitro without blocking ATP hydrolysis. Conclusions: Dynarrestin provides a novel dynein inhibitor with distinct specificity that uniquely facilitates probing dynein function. Given its multiple contributions to Hh signaling, mitosis and other cellular events, dynein remains a highly attractive target for medicinal chemistry programs aimed at exploiting and modulating its complex, poorly understood chemical cycle to interfere with different aspects of activity and function. We believe that unraveling dynein-dependent cellular processes by dynarrestin has great potential for development into novel anti-cancer drugs controlling Hh signaling downstream of Smoothened. Citation Format: Matthias Baumann, Susanne Höing, Ting-Yu Yeh, Nancy Martinez, Peter Habenberger, Lea Kremer, Hannes CA. Drexler, Philipp Küchler, Peter Reinhardt, Axel Choidas, Mia-Lisa Zischinsky, Gunther Zischinsky, Stephanie A. Ketcham, Lydia Wagner, Peter Nussbaumer, Slava Ziegler, Bert Klebl, Trina Schroer, Hans R. Schöler, Herbert Waldmann, Jared L. Sterneckert. Dynarrestin, a novel dynein inhibitor that does not block ciliogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2997.

Breast cancer is the second leading cause of cancer related deaths in women and more than 90% of these deaths result from metastatic disease rather than from the primary tumor burden. Recent studies have shown that intratumoral heterogeneity exists, and can affect chemotherapeutic and as well as metastatic outcome. To determine if crosstalk between metastatic and non-metastatic breast cancer cells can occur in the earlier stages of metastasis, where an epithelial-to-mesenchymal transition (EMT) is thought to occur in a small population of cells within a tumor, allowing them to metastasize, we examined whether different EMT-inducing pro-metastatic transcription factors (TFs) can influence the EMT status and metastatic properties of cells not expressing these factors (control cells). Twist1 and Snail1 are two EMT-inducing TFs that are known to increase metastasis through cell autonomous mechanisms. Our data demonstrate that these TFs can also non-cell autonomously alter the expression of numerous genes and proteins associated with EMT, as well as increase migration and invasion, when conditioned medium (CM) taken from HMLER-Twist1/Snail1 cells is placed on control cells. We show that the non-cell autonomous effects of Twist1 and Snail1 are in part induced via their ability to up-regulate another known EMT-TF, Six1. Loss of Six1 downstream of these TFs mitigates their ability to non-cell autonomously influence migration and invasion of non-metastatic, non-EMT TF expressing cells, indicating that Six1 is a key player downstream of Twist1 and Snail1. In multiple breast cancer models (including the MCF7 and 4t1 series of cell lines), CM taken from cells that overexpress Six1, when placed on control cells, leads to alterations in expression of EMT-related proteins, as well as increased migration and invasion. This does not occur when CM is taken from Six1 knockdown cells, showing that Six1 can non-cell autonomously cause an EMT in the control non-Six1 expressing cells. Using mouse models, we also demonstrate that highly metastatic, Six1 expressing cells can non-cell autonomously increase the metastatic incidence of weakly metastatic, non-Six1 expressing cells. We find that CM taken from cells expressing Twist1, Snail1, and Six1, when placed on control cells, leads to the induction of hedgehog (Hh) signaling as measured by Gli activity in the control recipient cells. Further, we demonstrate that activation of Hh signaling in control cells (when exposed to CM from metastatic-EMT cells) is critical for the induction of EMT-like characteristics. Interestingly, the mechanism of Gli activation in recipient cells differs depending on the EMT-TF expressed by the cells from which the CM is taken; such that upstream inhibitors of the pathway (cyclopamine and 5E1 monoclonal antibody against Hh ligands) only work in specific contexts, whereas more downstream inhibitors targeting Gli1 (Gant61) inhibit the effects in all contexts. These data suggest that EMT-TFs are able to activate the Gli signaling pathway non-cell autonomously via multiple mechanisms. We observe a strong correlation of Twist1, Snail1, and Six1 with Gli1 in numerous breast cancer datasets, whereas such a correlation is not observed with the ligands of Hh signaling, the more upstream activators of the pathway; an indication that the EMT-TFs may be converging in on increasing Gli activity and hence activating the Hh signaling pathway. Our data demonstrate that EMT-TFs, even if expressed in only a percentage of cells within a primary tumor, can increase metastasis of neighboring cells, and further suggest that inhibitors targeting downstream aspects of the Hedgehog pathway, such as Gli1, may be a novel means to target metastatic progression in heterogeneous breast cancers to increase patient survival. Citation Format: Deepika Neelakantan, Hengo Zhou, Joshua Cabrera, Heide Ford. EMT-inducing transcription factors Twist1, Snail1 and Six1 increase metastasis of neighboring tumor cells via induction of Hedgehog-Gli signaling. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B46.

RMS is the most common sarcoma of childhood. Approximately 60% of embryonal RMS (ERMS) and more rarely alveolar RMS (ARMS) express HH pathway components. However, roles for the HH signal transduction pathway in RMS biology and as a therapeutic target in RMS have been incompletely characterized. Therefore, we determined if RMS cells lines have HH signaling pathways that are responsive to pathway activation or inhibition in vitro. We demonstrated expression of HH pathway genes (Indian HH, Desert HH, PTCH1, SMO, GLI1, GLI2, and GLI3) by qRT PCR in ERMS (Rh18, Ruch2, SMS-CTR, and RD) and ARMS (Rh 30 and Rh41) cells. We showed high GLI1 protein expression, the downstream mediator of HH signaling, by Western blot in Rh18, Rh30, and Rh41 cells but not in RD or Rh28 cells. Exposure of Rh41 cells to Sonic HH-containing conditioned media caused a 12-fold increase in PTCH1 expression and exposure of Rh28 cells to Sonic HH peptide increased GLI1 expression 1.5-fold, suggesting intact HH signal transduction in some RMS cell lines. Down-regulation of HH pathway activity with the GLI1 inhibitor GANT61 reduced Rh41 and RD cell viability by MTT assays in a dose dependent manner. Reduced cell viability appeared to result at least in part from down-regulation of the anti-apoptotic GLI1 target gene, BCL2. These results suggest a role for HH signaling in RMS cell survival. Next, we used subcutaneous xenografts (with either Rh30 cells, n = 4; Rh41cells, n = 3; or RD cells, n = 3) to assess whether the HH pathway represents a potentially important therapeutic target in RMS in vivo. Mice were treated with either cyclopamine (subcutaneous SMO inhibitor), GANT61 (subcutaneous GLI1 inhibitor), or Vismodegib (oral SMO inhibitor) for 16 days once the tumor volume was 250 mm3. The study endpoints were HH pathway inhibition (qRT PCR), tumor volume (mm3), proliferation (BrdU immunostain), and apoptosis (Caspase immunostain) on day 17 of treatment. Cyclopamine and GANT61 reduced GLI1, PTCH1, and BCL2 expression in 7/10 and 4/10 mice, respectively. We observed partial responses to cyclopamine and GANT61 in Rh41 xenograft mice; GANT61 caused significant regression in tumor volume from baseline (p = 0.03). We did not detect changes in proliferation or apoptosis post-therapy by immunohistochemistry. Our results suggest that targeted inhibition of the HH signaling pathway with small-molecule inhibitors sometimes inhibits RMS xenograft tumor growth in vivo and support further RMS xenograft studies, assessing HH pathway inhibitors in combination with chemotherapeutic agents. Citation Format: Joon Won Yoon, Christopher Chandler, Marilyn Lamm, King-Fu Leong, Stephen Iannaccone, Gregory Taborn, Philip Iannaccone, David Walterhouse. Targeted inhibition of Hedgehog (HH) signal transduction in rhabdomyosarcoma (RMS) reduces cell survival in vitro and tumor growth in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3245. doi:10.1158/1538-7445.AM2015-3245

The ABC transporters, including ABCG2 or breast cancer resistance protein (BCRP) are drug efflux transporters. Their overexpression in cancers including breast cancer has been associated with the development of multidrug resistance (MDR) due to ABC transporter-mediated export from tumour cells of therapeutic drugs such as chemotherapeutic (eg. mitoxantrone, doxorubicin) and targeted agents (eg. tyrosine kinase inhibitors). PCR microarray analysis identified that ABCG2 expression was downregulated in the breast cancer cell lines, MCF-7 and T-47D following treatment of cells with the androgen, 5α-dihydrotestosterone (DHT), the Hedgehog pathway inhibitor, cyclopamine, or a combination of 10-8M DHT and 2μM cyclopamine. RT-qPCR analysis confirmed that DHT and cyclopamine treatments of MCF-7 cells rapidly downregulated ABCG2 mRNA levels by 24 hours. ABCG2 protein levels were also progressively decreased over 8 days of treatment, with earlier reductions in ABCG2 levels observed in cells co-treated with DHT and cyclopamine. As the ABC transporters are primarily active on membrane surfaces as drug exporters, the intracellular localisation of ABCG2 was investigated by immunofluorescence microscopy. In MCF-7 cells, DHT and/or cyclopamine decreased ABCG2 accumulation in cell-to-cell membrane junction complexes and interestingly, accumulation of ABCG2 into cytoplasmic vesicles which resembled aggresomes was observed in cells exposed to cyclopamine. Consistent with the reduced levels of membrane-bound ABCG2, export of the fluorescent ABCG2-specific substrate, mitoxantrone from MCF-7 cells was delayed following treatment of cultures with DHT and cyclopamine, thereby indicating diminished ABCG2 efflux activities. Moreover, these DHT- and cyclopamine-treated cells were also more responsive to the cytotoxic effects of mitoxantrone as the IC50 for mitoxantrone was decreased by ∼80% in treated cells compared to controls. Resistance to drug therapies, which is often facilitated by the ABC transporters, is a major cause of treatment failure and recurrence of breast cancer. As such, development and implementation of adjuvant compounds such as androgens and Hedgehog inhibitors, which inhibit expression and function of ABCG2 and other efflux transporters in breast cancer cells, may improve treatment outcomes and prolong recurrence-free survival of patients. Citation Format: Vivian YL Chua, Jennet Harvey, Jacqueline Bentel. Regulation of the ABCG2 drug efflux transporter in breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 715. doi:10.1158/1538-7445.AM2015-715

Drug profiling of cancer cell lines is a useful tool for predictive biomarker discovery, traditional done in two-dimensional cultures, a growing body of evidence suggests that results cannot always be translated into xenografts or in the clinic due to the lack of complexity of the culture conditions. 3D cultures offer the ability to carry out the same type of studies with the added benefit of incorporating the 3-dimentional cellular interactions and physiological gradients normally seen in a tumor microenvironment. In this study we used Numira's 3D cell line screening platform on Gefitinib, a selective inhibitor of epidermal growth factor receptor (EGFR). We determined IC50 of Gefitinib in a panel of 60 cell lines grown as spheroids and used this information to investigate genes associated with sensitivity. Two mutated genes were associated with sensitivity to Gefitinb, as expected, the epidermal growth factor receptor (EGFR) was one of them, since mutations on EGFR are required for clinical efficacy. The second gene was smoothened (SMO), a G protein-coupled receptor of the hedgehog signaling pathway. It has been suggested that blockade of the hedgehog signaling pathway enhances anti-proliferative effects of EGFR inhibitors, we explored this potential synergy in a combination studies of Gefitinib and SMO inhibitors, Cyclopamine and GDC0449, the results are discussed. Citation Format: Gonzalo Castillo, Xiuyun Jiang. Association of the hedgehog signal pathway to Gefitinib sensitivity in a 3D cancer cell lines screening. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5447. doi:10.1158/1538-7445.AM2015-5447

Adults diagnosed with glioblastoma multiforme (GBM) typically have a median survival less than 15 months. GBM patient biopsies and derived cell lines contain subsets of cells extending primary cilia, microtubular antennae that mediate signaling events reported to influence cell proliferation and tumor cell growth. However, it remains unclear how cilia contribute to the phenotypic characteristics of GBM. To determine whether cilia influence GBM tumor progression, we generated several stable patient-derived cell lines devoid of cilia by expressing a dominant-negative form of Kif3a (dnKif3a), a protein normally essential for ciliogenesis. Depending on the line, mice receiving intracranial implants of dnKif3a-expressing cells survived shorter, as long as, or significantly longer than mice implanted with control cells. To determine if ciliated cells derived from different GBM tumors share similar sensitivities to sonic hedgehog (SHH), a mitogen present in adult brain that activates receptor signaling cascades within cilia, we exposed control and dnKif3a-expressing cells in vitro to vehicle or recombinant SHH. Interestingly, only the cell line that prolonged survival in vivo displayed significant increases in proliferation following exposure to SHH, an effect that was blocked by either dnKif3a or pretreatment of the cells with cyclopamine, an inhibitor of the SHH cascade. All cell lines tested appeared to respond to SHH as evidenced by activation of downstream SHH signaling components in their cilia. Collectively, our data suggest that strategies to disrupt primary cilia in GBM patients could lead to inhibition or enhancement of tumor growth potentially by altering cell sensitivity to the extracellular environment. Citation Format: Lan Hoang-Minh, Dorit Siebzehnrubl, Loic Deleyrolle, George Ugartemendia, Hunter Futch, Benjamin Griffith, Joshua Breunig, Susan Semple-Rowland, Brent Reynolds, Matthew Sarkisian. Targeting KIF3a and primary cilia differentially affects sonic hedgehog sensitivity and the rate of glioblastoma progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1542. doi:10.1158/1538-7445.AM2015-1542

Neuroblastoma and medulloblastoma are malignant solid tumors of childhood and early adolescence, that often reappear after clinical treatment due of an acquired chemo-resistance. These tumors present an increased expression of the Sonic Hedgehog signaling pathway. In the last years, inhibitors of this pathway, such as cyclopamine are being studied as a possible alternative to current treatment. In this research, we aim at analyzing the appearance and characteristics of resistance derived from the treatment with mutation-selective concentrations of cyclopamine. The aim of this study is the design of different ways to bypass the resistance generated by the tumor once its biology is completely understood. Cells were exposed to several treatment cycles. A 24 h cyclopamine exposure was performed when cell culture reached 60-70% confluence. Basal half-maximal inhibitory concentration (IC50) was 10 μM. Hence, SK-N-DZ cells received 5 treatment cycles of cyclopamine at 12 μM dissolved in DMSO; whereas Daoy cells received 5 treatment cycles of cyclopamine at 10 μM. The control group only received the diluent. Anchorage-independent growth assays (in soft agar), clonogenicity assays, cell migration scratch (wound-healing) assays, and relative mRNA expression were studied during cycles 1-5. During the first 5 treatment cycles, mRNA expression of SMO remained constant. CD15 expression decreased. MYCN, Gli and expression showed an increasing trend. Decrease in the pro-apoptotic status was also described. Soft agar showed an increase tendency in relative colony formation capacity. No statistical differences were found in the clonogenicity assays for the SK-ND-Z cells. The results highlighted the evidence that cells became resistant because they were able to grow in soft agar without attachment, to migrate and to overexpress genes related to cancer stem cells. Results shown are congruent with a resistance generation possibly owing to multidrug efflux pumps. However, more clear results would be seen with 48 h treatment. Further analysis based on this work could include 48 h treatment, water-dissolvable cyclopamine performance study, a more complete gene analysis, or studies with other neurological tumors. Note: This abstract was not presented at the meeting. Citation Format: Javier de la Rosa, Amaia Paredes, Javier Asensio-Salazar, Xing Fan, Bárbara Meléndez, Juan A. Rey, Javier S. Castresana. Induction and characterization of cyclopamine resistance in SK-N-DZ neuroblastoma and DAOY medulloblastoma cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3282. doi:10.1158/1538-7445.AM2015-3282

Introduction: The sonic hedgehog (SHH) signaling pathway regulates embryonic developmental processes such as pattern formation, differentiation, proliferation, and organogenesis and has been hypothesized to play an integral role in the maintenance and progression of bladder cancer. Blocking this pathway may be an efficacious molecular target against high-risk bladder cancer. Methods: SHH pathway protein expression was assessed in an in-house human bladder cancer tissue array comprising superficial, invasive and lymph node metastasized transitional cell carcinoma. In addition, a panel of bladder cancer cell lines was assessed for SHH pathway activation and two lines, UM-UC3 and 253J-BV, were further evaluated for SHH pathway function and responsiveness. Messenger RNA and protein expression, proliferation, apoptosis and cell invasion were measured in response to the addition of SHH ligand, a SHH overexpressing construct, ligand and receptor blockers, small-molecule pathway inhibitors, and antisense oligonucleotides (ASO) targeted to the mRNA sequence of the down-stream transcription factors Gli family zinc fingers 1 and 2 (Gli1, Gli2). In-vitro ASO results were confirmed in an in-vivo experiment on UM-UC3 cells intravesically instilled into the bladder of nude mice. Results: Bladder tissues show a positive and variable pattern of SHH pathway expression within and between tumors. The bladder cancer cell lines UM-UC3 and 253J-BV both express the main components of the SHH pathway, notably the transcription factors GLi1 and Gli2. The most effective inhibitors of UM-UC3 are an indirect ciliagenesis inhibitor and direct Gli knock-down through ASO treatment. Both cell lines show decreased growth and increased apoptosis with the smoothened protein inhibitor cyclopamine but 253J-BV does not respond in proliferation or apoptosis to Gli1 ASO suggesting a difference in pathway function between the lines. UM-UC3 secretes approximately 10 fold higher amounts of SHH than 253J-BV but does not respond to exogenous SHH treatment. 253J-BV does respond with an increase in proliferation, invasion, and SHH and Forkhead Box M1 expression. Robotnikinin and the SHH antibody 5E1 show a modest decrease in cell viability at high concentrations in both lines. The addition of SHH through plasmid transfection has no effect. In-vivo, Gli2 ASO significantly decreases UM-UC3 tumour growth. Conclusions: Bladder cancer cells are differentially sensitive to specific regions of SHH pathway modulation, as seen by a greater reliance on GLi2 in 253J-BV. Direct knock-down of the down-stream transcription factors Gli1 and Gli2 with ASOs is the most effective strategy and may be a promising treatment for bladder cancer. Citation Format: Peter A. Raven, Summer Lysakowski, Yoshiyuki Matsui, Shintaro Narita, Alan I. So. The sonic hedgehog pathway as a therapeutic target in bladder cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1510. doi:10.1158/1538-7445.AM2015-1510

The olfactomedin 4 gene (OLFM4) plays critical tumor suppressor roles in human and murine prostate cancer. We have previously reported that loss or reduce of OLFM4 expression is associated with the progression of human prostate cancer. We further found that loss of Olfm4 leads to prostate neoplastic progression in the Olfm4-knockout mouse model. To study the cellular and molecular mechanisms underlying neoplastic progression of the prostate, we have performed gene chip microarray and quantitative real-time RT-PCR analysis to compare Olfm4 knockout with wild-type mouse prostate. The gene-expression signature of prostate tissues from Olfm4-deficient mice revealed significant changes for genes associated with neoplastic progression. In this study, we further detected that hedgehog signaling-pathway components are up-regulated in Olfm4-knockout mice. Since hedgehog signaling pathways are involved in regulating prostate stem/ progenitor cell proliferation, we performed sphere formation analysis with Olfm4 mutant mouse prostate and found that loss of Olfm4 increased proliferative abilities of prostate stem/progenitor cells. Interestingly, administration of human OLFM4 protein and cyclopamine, a specific inhibitor of Shh-signaling pathway, into the sphere culture media reduced Olfm4-deficient mouse prostate stem/progenitor cell growth. To extend the observation from the mouse model, we established OLFM4-GFP fusion stably expressing cell clones by using human prostate cancer cell lines PC-3, 22RV1 and DU145. We found significant inhibition of sphere and colony formation in OLFM4 expressing cells compared with vector expressing control cells. The expression levels of Shh signaling pathway components were significantly inhibited in OLFM4 expression cells compared to vector expressing control cells. To detect whether or not OLFM4 down regulates the Shh signaling pathway through interaction with Shh protein, we performed co-immunopricipitation in 293T and PC-3 OLFM4 stably expressing cells. We found that OLFM4 directly interacts with Shh protein. We then carried out Gli- reporter assay using 293T and prostate cancer cells. We demonstrated that expression of OLFM4 inhibited Gli-reporter activity in a dose-dependent manner. Taken together, these results suggest that OLFM4 inhibits prostate stem/progenitor cells growth by mediating the Shh signaling pathway through a direct interaction of OLFM4 with Shh protein. Citation Format: Hongzhen Li, Wenli Liu, Weiping Chen, Jianqiong Zhu, Chu-Xia Deng, Griffin P. Rodgers. Olfactomedin 4 inhibits prostate stem/progenitor cells growth through regulating the hedgehog signaling pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 604. doi:10.1158/1538-7445.AM2014-604

Accumulating evidences have demonstrated the antineoplastic properties of sulforaphane, a major isothiocyanates present in cruciferous vegetables. However, the natural analogue of sulforaphane, sulforaphene, rich in radish, has yet not been extensively studied for its potential in preventing cancer. In this study, sulforaphene exerted inhibiting effect on migration of SUM159 human breast cancer cells, the migrated cells were reduced from 72.3% in control group to 17.5% in sulforaphene(10uM) treated group. The Hedgehog signaling, known to be involved in cancer invasiveness, was found to be regulated by sulforaphene. The mRNA and protein expression of Gli1 and Smoothened(Smo) which were Hedgehog signaling activating mediators were significantly suppressed by sulforaphene, but the expression of Patched1(Ptch1) an inhibiting mediator, was induced. In addition, the Hedgehog signaling inhibitors, cyclopamine and vismodegib, strongly inhibited the migration of SUM159 breast cancer cells, suggesting the association of Hedgehog signaling with the migration of breast cancer cells. Taken together, sulforaphene significantly inhibited the migration via down-regulating the Hedgehog signaling pathway and suggest that sulforaphene may be considered as a potent phytochemical in suppressing the mammary carcinogenesis. Citation Format: Cheng Bao, Jaehoo Lee, Jiwon Ko, Hyun-Chang Park, Hong jin Lee. Sulforaphene inhibited migration through down-regulating the Hedgehog signaling in SUM159 human breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 121. doi:10.1158/1538-7445.AM2014-121

Metastatic cancer cells possess a unique capacity to survive without appropriate signaling from the extracellular matrix (ECM). The epithelial cells surrounded by proper ECM within tissues are designed to grow and differentiate whereas epithelial cells lacking suitable EMC are destined to anoikis, a form of apoptosis. The metastatic cancer cells resist anoikis to survive under the absence of ECM. Current study identifies a novel interplay between HER2 and sonic hedgehog signaling in promoting anoikis resistance. MDA-MB-231 cells exhibited an up-regulation of HER2 expression in cells cultured under anchorage- independent conditions as compared to adherent cells. Similar phenomenon was also observed in 4T-1 cells. The survival of MDA-MB-231 cells with stable HER2 overexpression (HH) was significantly enhanced when cultured under anchorage-independent conditions. However, the enhanced survival of MDA-MB-231 (HH) was reduced when HER2 was silenced using shRNA. These observations indicate the obligatory role of HER2 in anoikis resistance. Furthermore cells grown under anchorage-independent conditions induced the expression of sonic hedgehog (SHH). Increase in SHH was accompanied with enhanced expressions of PTCH2 and GLI2, the receptor and subsequent transcription factors of the sonic hedgehog signaling. Our results further showed that the induction of SHH was significantly higher in MDA-MB-231 (HH) cells relative to parent cells when cultured under anchorage-independent conditions, suggesting a correlation between HER2 and SHH signaling. Treatment of MDA-MB-231 and 4T-1 cells with cyclopamine (SHH signaling inhibitor) reduced the survival of cells under anchorage-independent conditions. Interestingly, cyclopamine treatment also inhibited the induction of HER2 during anchorage-independent culture conditions. Taken together, our study suggests a SHH-mediated induction of HER2 to promote anoikis resistance in breast cancer cells. Further mechanistic and in vivo studies are under progress. [Supported in part by R01 grant CA129038 (to S.K.S) awarded by the National Cancer Institute]. Citation Format: Parul Gupta, Sanjay K. Srivastava. A unique cross-talk between HER2 and sonic hedgehog signaling promotes anoikis resistance in breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1966. doi:10.1158/1538-7445.AM2014-1966

Hedgehog (Hh) signaling is a highly conserved inter- and intracellular communication mechanism that governs organogenesis and is dysregulated in cancers of numerous tissues, including prostate. Up-regulated expression of the Hh ligands, Sonic (Shh) and Desert (Dhh), has been reported in androgen-deprived and castration-resistant prostate cancer (CRPC). In a cohort of therapy naive, short- and long-term neoadjuvant hormone therapy-treated (NHT), and CRPC specimens, we observed elevated Dhh expression predominantly in long-term NHT specimens and elevated Shh expression predominantly in CRPC specimens. Together with previously demonstrated reciprocal signaling between Shh-producing prostate cancer (PCa) cells and urogenital mesenchymal fibroblasts, these results suggest that castration-induced Hh expression promotes CRPC progression through reciprocal paracrine signaling within the tumor microenvironment. We tested whether the orally available Smoothened (Smo) antagonist, TAK-441, could impair castration-resistant progression of LNCaP PCa xenografts by disrupting paracrine Hh signaling. While TAK-441 or cyclopamine did not affect androgen withdrawal-induced Shh up-regulation or viability of LNCaP cells, castration-resistant progression of LNCaP xenografts was significantly delayed in animals treated with TAK-441. In TAK-441-treated xenografts, expression of murine orthologues of the Hh-activated genes, Gli1, Gli2, and Ptch1, was substantially suppressed, while expression of the corresponding human orthologues was unaffected. Since androgen-deprived LNCaP cells up-regulate Shh expression, but are not sensitive to Smo antagonists, these studies indicate that TAK-441 leads to delayed castration-resistant progression of LNCaP xenografts by disrupting paracrine Hh signaling with the tumor stroma. Thus, paracrine Hh signaling may offer unique opportunities for prognostic biomarker development, drug targeting and therapeutic response monitoring of PCa progression. Citation Format: Mazyar Ghaffari, Naokazu Ibuki, Irene Iu, Mitali Pandey, Ladan Fazli, Masahide Kashiwagi, Hideaki Tojo, Osamu Nakanishi, Martin E. Gleave, Michael E. Cox. A novel smoothened antagonist, TAK-441, delays castration-resistant progression in prostate cancer by disrupting paracrine hedgehog signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4952. doi:10.1158/1538-7445.AM2013-4952

Introduction: Prostate cancer (PCa) is the second most common cause of death from cancer and the leading cancer diagnosed in the US men. Approximately 1 in 6 American men will be diagnosed with prostate cancer during his lifetime. Estimated new cases and deaths from prostate cancer in the United States for 2012 include: 241,740 new cases and 28,710 deaths. Initial treatment for metastatic PCa involves androgen ablation therapy, which causes regression of androgen-dependent tumors. However they all eventually relapse resulting in recurrent androgen independent prostate cancer (AIPC). Methods: This study aims at assessing the role of Hedgehog (Hh) and Androgen receptor (AR) signaling in the development of AIPC. Quantitative RT-PCR analysis was performed for AIPC cells in vitro and for AIPC xenograft after castration, to assess the levels of Hh and AR signaling components. Hh inhibitors, LDE225 (Novartis) and Cyclopamine, and Androgen receptor inhibitor Pyrvinium pamoate were tested individually and in combination to assess the suppression of AIPC progression both in vitro and in vivo. AR-responsive and Gli-responsive promoter-luciferase assay and MTT assay were used for in vitro studies. Intraperitoneal administration of drugs and bioluminescence imaging for monitoring orthotopic tumors in mice were used to study the effect of drugs on the growth of AIPC in vivo. Results: We demonstrate that androgen deprivation leads to increase in Hedgehog (Hh) signaling components in the AIPC cells in vitro and in vivo. AIPC cells also show increased sensitivity to low concentrations of androgen, suggesting increased androgen receptor expression. Together, these results suggest that increased Hh signaling and androgen receptor expression in the absence of androgen agonist plays an important role in the development and progression of AIPC. Our results show that inhibiting either Hh or androgen receptor signaling pathway individually was not effective in preventing AIPC cell proliferation in vitro as well as in inhibiting xenograft tumor growth in mice. In contrast, combination of an Hh pathway inhibitor with an androgen receptor inhibitor synergistically suppressed the growth of AIPC cells in vitro as assessed by MTT assay and reduced tumor burden in a mouse xenograft model of AIPC as compared to individual treatments.Conclusion: Upregulated Hh signaling and AR expression mediates development and progression of AIPC. Combined inhibition of Hh and androgen receptor signaling pathways may be a novel effective therapeutic strategy for the treatment of androgen independent prostate cancer. Citation Format: pramod gowda, Jianhong Deng, Sweta Mishra, LuZhe Sun. Inhibition of hedgehog and androgen receptor signaling pathways produced synergistic suppression of androgen independent prostate cancer progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2126. doi:10.1158/1538-7445.AM2013-2126

In the US, about 10-20% of breast cancers are found to be triple-negative breast cancer(TNBC). Because of the lack of estrogen receptor (ER) and progesterone receptor (PR) expression, and human epidermal growth factor receptor 2 (HER2) amplification, TNBC does not benefit from the currently available targeted therapies. Furthermore, recent molecular analysis of TNBC cell lines and lesions has suggested that these tumors might be enriched with cancer initiating cells (CICs). This possibility is consistent with the failure of conventional therapies to control disease progression, because according to the cancer stem cell theory, CICs play a major role in disease recurrence and metastatic spread, the two major causes of patient mortality. These findings and the lack of curative therapy for any TNBC patient with metastatic disease emphasize the need to develop effective novel therapies for this type of breast cancer. To address this need, we have developed a combinatorial immunotherapy, which eliminates both differentiated TNBC cells and TNBC CICs. The tumor antigen we have selected as a target is chondroitin sulfate proteoglycan (CSPG)4, since this membrane bound tumor antigen is expressed at high levels on both differentiated TNBC cells and TNBC CICs. CSPG4 plays a crucial role in the biology of TNBC cells, since it is involved in ERK and FAK signaling pathways which are associated with TNBC cell proliferation and migration. Furthermore, it is upregulated on activated pericytes in the tumor microenvironment. CSPG4-specific mAb 225.28 inhibits TNBC cell growth and migration. Moreover, it inhibits signaling pathways associated with TNBC cell growth and survival. It also can selectively inhibit neoangiogenesis in the tumor microenvironment, contributing to the elimination of TNBC cells, even those that do not express CSPG4. To enhance the targeting of TNBC CICs, immunotherapy is combined with the administration of an inhibitor (cyclopamine/ LDE225) of the Sonic Hedgehog Homolog (SHH) pathway which is activated in TNBC cells and, especially in TNBC CICs. Inhibition of TNBC CIC growth by mAb 225.28 is around 10%, but is enhanced up to about 70% when mAb 225.28 is combined with a SHH pathway inhibitor. Our in vitro findings strongly suggest that targeting CSPG4 with mAb 225.28 in combination with a SHH inhibitor may represent an effective strategy to eliminate differentiated TNBC cells and TNBC CICs. Citation Format: Yangyang Wang, Francesco Sabbatino, Soldano Ferrone, Xinhui Wang. Inhibition of TNBC cell growth by CSPG4-specific mAb 225.28 with a Sonic Hedgehog pathway inhibitor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4741. doi:10.1158/1538-7445.AM2013-4741

Background: Cancer stem cells (CSCs) have unique functions of self-renewal, pluripotency and hierarchical proliferation. CSCs are considered to play a crucial role in the recurrence of cancer and are assumed resistance to anticancer agents. Aberrant Hedgehog (Hh) pathway signaling has been implicated in the pathogenesis, self-renewal, and drug resistance of a growing number of solid malignancies. Sonic hedgehog (Shh) is expressed in PanIN and pancreatic adenocarcinoma, especially implicating as an early and late mediator of pancreatic cancer tumorigenesis. In this study, we focused on Hh pathway to regulate self-renewal of CD133high pancreatic CSCs. Materials and Methods:Cells: We established endogenous CD133high (over 90%) pancreatic cancer cells derived from Capan-1 cells. Consequently, CD133knock-down cells were established by the lentiviral transduction method. Reagents: 1) Cyclopamine inhibits the Hh pathway by interaction with the Hh signaling protein smoothened (SMO). 2) GANT61 blocks the GLI function which constitutes the final step in the Hh pathway and interferes with GLI-1 DNA binding in living cells. To analyze the Hh pathway: Stemness-related genes were analyzed by DNA microarray. Self-renewal signal activation was confirmed by fluorescent immunohistochemistry. To analyze Hh pathway, sphere assay and MTT assay were performed on sphere-forming abilities and cytostatic effects by Hh signal inhibitors, respectively. The migration assay was used to evaluate cell migratory ability. Results: 1) Sphere-forming cells in CD133high cells showed upregulation of BMP3, SOX2, GLI1, ALDH1A, et al. and downregulation of PTCH1, SMO, WNT3A, et al. by DNA microarray but the monolayer cells did not. 2) Cyclopamine could suppress cell growth and sphere formation of CD133high and CD133knock-down cells. GANT61 showed more suppressive effect than Cyclopamine. In addition, GANT61 treatment showed smaller spheroid than the cyclopamine treatment. These results suggest that Hh signal participates in proliferation and self-renewal of the pancreatic CSCs. 3) CD133high cells showed higher migratory ability than CD133knock-down cells. Because the expression of epithelial-mesenchymal transition (EMT)-associated transcription factors, such as Slug and snail, in CD133high cells are upregulated but not in CD133knock-down cells. Interestingly, Hh signal inhibitors suppressed the migratory ability of CD133high cells by the migration assay. Conclusion: GLI-1 acts more effectively on self-renewal of pancreatic CSCs through Hh pathway than Cyclopamine. Further study on GLI-1 inhibitor can lead to a new targeted therapy for invasion and metastasis of pancreatic cancer. Citation Format: Yumi Miyazaki, Qiang Ding, Makoto Yoshimitsu, Toru Obara, Koichirou Tsukasa, Shyuichiro Matsubara, Sonshin Takao. GLI-1 plays a pivotal role in Hedgehog pathway on self-renewal of pancreatic cancer stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 214. doi:10.1158/1538-7445.AM2013-214

The transcription factor Activator Protein 1 (AP1), is an important regulator in cisplatin resistance. AP1 is comprised of the two proteins, c-jun and c-fos. The transcription factor Gli1, a member of the hedgehog signaling pathway, is also an important factor in cisplatin resistance and transcriptionally regulated c-jun. The c-jun interface with Gli1 is mediated by a 130 kDa isoform of Gli1. We have investigated whether Gli1 may regulate c-fos by selectively knocking down Gli1 expression using anti-Gli1 shRNA or by treating cells with a Hedgehog pathway inhibitor, cyclopamine. A2780-CP70 cells were treated with an IC50 dose of anti-Gli1 shRNA and the changes in RNA and protein levels were analyzed over a 72 hour time course. No changes in the expression of c-jun or c-fos RNA transcripts were observed versus control. Western blot analyses showed Gli1 protein expression was reduced at 24 hours and undetectable at 48 and 72 hours. Sonic hedgehog (Shh) protein levels were dramatically reduced by 24 hours, low but measureable at 48 hours, and undetectable at 72 hours. Indian hedgehog (Ihh) expression increased at 24 hours and remained stable for the remainder of the 72 hour time course. Treatment of A2780-CP70 cells with an IC50 dose of cyclopamine resulted in a different intracellular response than treatment with Gli1 shRNA. Cyclopamine specifically targets the cell membrane receptor Smoothened. Experiments were performed analyzing RNA and protein expression over a 72 hour time period after treatment with cyclopamine. C-jun and c-fos RNA transcript levels were higher than control 6 hours after treatment and remained high for the duration of the 72 hour experiment. Western blot analyses after cyclopamine treatment showed Gli1 protein expression gradually decreasing during the 72 hours, but were never fully depleted. Expression of Shh increased over 72 hours, while Ihh levels remained stable. The identical Gli-Binding-Site (GBS) in the c-jun promoter was found in the promoter of c-fos. We investigated whether the Gli1 isoform that binds c-jun, also binds c-fos. Simultaneous Western and Southwestern blots show only one of the known Gli1 isoform binds the promoter of c-fos. Further studies with Chromatin Immunoprecipitation (ChIP) assays confirmed the 130 kDa isoform of Gli1 binds the GBS of the c-fos promoter. We conclude that the 130 kDa Gli1 isoform binds both components of AP1, c-jun and c-fos, and modulates the cellular resistance to cisplatin. Citation Format: Lauren Amable, Kenji Kudo, Elaine Gavin, Jason Fain, Eddie Reed. A specific Isoform of Gli1 binds the promoter region of c-fos in A2780-CP70 human ovarian cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-223. doi:10.1158/1538-7445.AM2013-LB-223

Recurrence of breast cancer after chemotherapy is thought to arise from resistant breast cancer stem cells which are eventually able to repopulate the tumor. The hedgehog (HH) signaling pathway has been shown to regulate the proliferation and survival of breast cancer stem cells, and has been shown to promote resistance to chemotherapy through the activation of multi-drug resistance and prosurvival pathways. Here we report that exposure of heterogenous breast cancer cell lines (T47D and SKBr3) to low doses (1-10nM) of docetaxel resulted in activation of HH signaling. Increased levels of sonic HH were detected in supernatants of breast cancer cells at 6- hours post-treatment. This release was accompanied by increased expression and nuclear translocation of Gli-1, peaking at 24 hours after exposure. Additionally, increased expression of the PTCH receptor was observed by western blotting in these cells 24 hours after treatment, indicating activation of a Gli-1 induced negative feeback loop. Increased mammosphere formation was observed in cells after preincubation with low dose docetaxel. Secondary sphere forming efficiency (SFE) was also increased in pretreated cells. The increase in SFE was similar to that of cell lines cultured in the presence of recombinant SHH. Additionally, the increase in SFE observed after docetaxel treatment could be blocked by the addition of sublethal doses of the hedgehog inhibitor cyclopamine. These results suggest that hedgehog pathway activation induced by docetaxel treatment could result in increased stemness of breast cancer cell lines. We propose that chemotherapy may activate stem cell signaling pathways, resulting in expansion of cancer stem cells and increased drug resistance. Citation Format: Lynn Opdenaker, Jennifer Sims-Mourtada. Taxane-induced hedgehog activation promotes stemness of breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3718. doi:10.1158/1538-7445.AM2013-3718

Medulloblastoma is one of the most frequent and aggressive tumors of childhood. The Sonic hedgehog (Shh) pathway, related to human development, is altered in most medulloblastomas: genes like Ptch, Smo, or Sufu suffer mutations in 15% to 25% of these tumors. We tested Shh inhibition in the Daoy medulloblastoma cell line by two methods: a molecular one (direct Gli1 siRNA inhibition); and a pharmacological inhibition of Smo, upstream of Gli1, by cyclopamine. Afterwards, a comparison of cellular and molecular responses was done. We proved that MTT cell viability, and cell migration assessed by the scratching assay decreased after Shh inhibition. Furthermore, colony formation assay in culture decreased by 70%, and colony formation assay in soft agar decreased up to 90% when Shh inhibition was applied. As a whole, Shh inhibition conferred a less in vitro tumorigenic status to Daoy cells. Moreover, we assessed the expression of different Gli1 target genes and other genes, before and after Shh inhibition, and found that Shh shows a crosstalk with oncogenes and tumor suppressor genes that have been described in numerous tumors. Therefore, we found downregulation of Ptch1, Cyclin D2, Plakoglobin, Nkx2.2, Bmi1, Smo and N-myc after Shh inhibition. Sufu and Gli3 showed parallel results, where Gli1 siRNA did neither decrease nor increase expression of both genes, whereas cyclopamine reduced them in 15-25%. Pax6 mRNA levels were upregulated by either Gli1 siRNA or cyclopamine. Finally, Notch1 was upregulated after inhibition of Shh, while Notch2 showed contrasting results, as siRNA inhibition decreased its expression, while cyclopamine increased it. All these experiments give an overview of the Shh pathway in medulloblastoma, its relationship with other genes, and the demonstration of the efficacy of cyclopamine and Gli 1 siRNA Shh inhibition in vitro. Citation Format: R Garcia-Lopez, B Vera-Cano, A Vacas-Oleas, J de la Rosa, G Gallo-Oller, M H. Shahi, X Fan, M M. Alonso, J A. Rey, Javier S. Castresana. Sonic hedgehog inhibition reduces in vitro tumorigenesis and alters expression of GLI1-target genes in a desmoplastic medulloblastoma cell line. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5053. doi:10.1158/1538-7445.AM2013-5053

In spite of the decreasing lung cancer incidence recently observed in some countries, its mortality has remained high due to late presentation and limited chemotherapy effectiveness. Even for EGFR mutant cancers initially responding to tyrosine kinase inhibitors (TKI), long term patient survival has not been achieved due to the almost universal occurrence of TKI resistance. Tumor initiating cells (TIC) are a subpopulation of cancer cells believed to be capable of self-renewal leading to tumor perpetuation, metastasis and treatment resistance. To identify self-renewing cells in lung cancer and investigate their regulatory pathways, tumor cells with differential expression of the combined CD44 and ALDH1 markers were isolated by flow cytometry for functional comparison. The results showed in established lung cancer cell lines and early passage clinical lung cancer cells, the CD44high/ALDHhigh population had the strongest, while CD44low/ALDHlow the weakest in-vitro cell sphere formation and in-vivo serial xenograft transplantation capacities. The CD44high/ALDHhigh population also showed higher matrigel invasion ability, pluripotency genes expression, cell cycle regulator expression, as well as resistance to tyrosine kinase inhibitor and/or cisplatin treatment. TKI treatment led to increased CD44high/ALDHhigh cell proportions. Simultaneous down-regulation of CD44 by si-RNA and enzymatic inhibition of ALDH1 suppressed stem/progenitor genes expression including OCT4, SOX2 and NANOG, and attenuated cell sensitivity to TKI and cisplatin treatment in different lung cancer cells, indicating CD44 and ALDH were not only surface markers for TIC, but also had functional roles in TIC maintenance. To investigate for phenotypic differences and potential regulatory pathways involved in the maintenance of the two cell populations, the expression microarray profiles of isolated TIC (CD44high/ALDHhigh) and non-TIC (CD44low/ALDHlow) populations were compared. The results showed differential expression of various cell adhesion molecules, HLA markers, and signaling proteins involved in the Sonic Hedgehog (e.g. GLI1, GLI3) and Notch (e.g. DLL4, JAG1) pathways. Differential expression of selective molecules was independently validated by quantitative RT-PCR in an expanded panel of lung cancer cell lines. Pharmacological inhibition of the Hedgehog pathway using cyclopamine decreased TIC proportion in a dose dependent manner and sensitized lung cancer cells to TKI treatment, while Notch inhibition led to less prominent effects. Taken together, these findings indicated that CD44high/ALDHhigh expression characterized a subpopulation of self-renewing and tumorigenic cells in lung cancer. Therapeutic targeting of the Hedgehog pathway might be useful for disrupting TIC and help to achieve lung cancer control. Citation Format: Maria Pik Wong, Jing Liu, Zhi-jie Xiao. The CD44high/ALDHhigh cell population mediates tumor initiating cell properties through the sonic Hedgehog pathway in lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-271. doi:10.1158/1538-7445.AM2013-LB-271

Background: Neuroblastoma originates from neural crest cells and is one of the most common and deadly tumors of childhood. The majority of tumors develop therapy resistance with fatal clinical outcome despite intensive multimodal therapy. The Hedgehog (HH) signaling pathway is fundamental in embryonic development, controlling cell fate and proliferation. Activation of HH signaling is involved in several cancers, among them pediatric cancers. Studies have revealed that human neuroblastoma cells express high levels of proteins involved in HH signaling. GANT61 was recently identified as an inhibitor of GLI1 and GLI2, important transcription factors in the HH signaling pathway. The aim of this study was to investigate the impact of antagonists of HH signaling in neuroblastoma through characterization of the effects of small molecule inhibitors of the HH pathway, in vitro and in vivo. Methods: Cytotoxic activity of HH inhibitors was studied in cell viability and clonogenic assays. The molecular mechanisms were characterized using cell- and molecular biology techniques. Xenograft studies in mice were performed to validate the therapeutic effects and toxicity in vivo. Results: The cytotoxicity of different HH inhibitors (cyclopamine, SANT1 and GANT61) was evaluated in a panel of human neuroblastoma cell lines. GANT61 was the most potent drug tested with IC50 values in the range of 5.8 - 13 µM after 72h incubation. Expression of GLI1 significantly correlated to the log IC50 for GANT61 (R2=0.88), the higher the expression of GLI1 the lower the amount of GANT61 was required to inhibit cellular growth. We also saw an inverse correlation with MYCN (R2=−0.88). When measured with a GLI-dependent luciferase reporter plasmid, the GLI activity was decreased with 36% to 80% after GANT61 treatment (10 µM, 48h) depending on cell line. Investigation of cell cycle regulation and cell death demonstrated that GANT61 inhibited proliferation and induced apoptosis. siRNA experiments revealed that knockdown of GLI1 reduced proliferation more potently than GLI2, GLI3 and SMO inhibition. GANT61 significantly inhibited tumor growth in neuroblastoma xenografts in nude NMRI nu/nu mice without causing any sign of toxicity. Conclusions: These results suggest that inhibitors of the HH signaling pathway such as GANT61 may constitute a novel option for treatment to patient with high-risk neuroblastoma without MYCN-amplification. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4725. doi:1538-7445.AM2012-4725

Pancreatic cancer is among the deadliest of cancers as it is rarely curable and the 5-year survival rate of patients with any stage of the disease is approximately 6%. This dismal clinical picture is reflective of the poor response of this disease to chemo- and radio- therapy, as well as to surgery as conventionally used. The unmet clinical needs for patients with pancreatic cancer highlight the urgency to develop effective novel therapies for this disease. To address this need, we have developed a combinatorial immunotherapy which eliminates differentiated pancreatic adenocarcinoma cells and pancreatic cancer initiating cells (CICs). According to the cancer stem cell theory, CICs are responsible for disease recurrence and metastases. Therefore, they have to be eradicated in order to “cure” a malignant disease. The tumor antigen we have selected as a target is the HSP glucose-regulated protein of 94 kDa (Grp94), a member of the HSP90 family, since it plays an important role in the biology of malignant cells. Furthermore, we have recently developed a human monoclonal antibody (mAb), named mAb W9, which has a unique specificity. It recognizes an extracellular epitope of Grp94, which is expressed on many types of malignant cells, including differentiated pancreatic adenocarcinoma cells and pancreatic CICs. mAb W9 inhibited in vitro growth of the human pancreatic cancer cell lines PANC2.03 and PANC3.27 by ∼40%. The antiproliferative activity was increased to 90% when mAb W9 was combined with cyclopamine and 5-FU, both of which inhibit the hedgehog signaling pathway that is aberrantly activated in pancreatic adenocarcinoma, particularly in pancreatic CIC. The antitumor activity of mAb W9 reflected a decreased level of RAS, C-Raf, phosphorylated (p)-MEK, p-ERK, and p-AKT. mAb W9 had also an effect on pancreatic CICs, identified as cells with high aldehyde dehydrogenase expression. These cells are referred to as ALDHbright cells. Treatment of the human pancreatic cancer cell line MIA PaCa-2 in vitro with mAb W9, cyclopamine and 5-FU inhibited the growth of ALDHbright cells by ∼90%. In contrast, the inhibition was only 50% when cells were treated with mAb W9 or cyclopamine individually, 20% when they were treated with 5-FU and 70% when they were treated with mAb W9 in combination with cyclopamine or 5-FU. Apoptosis was induced in 70% of pancreatic cancer cells PANC1 by mAb W9 in combination with cyclopamine and 5-FU, but in less than 35% by the agents individually and by the combinations of any two of the agents. Our in vitro findings strongly suggest that targeting cell surface Grp94 with mAb W9 in combination with cyclopamine and 5-FU may represent an effective strategy to eliminate differentiated pancreatic cancer cells and pancreatic CICs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4390. doi:1538-7445.AM2012-4390

Sonic hedgehog signaling pathway plays an important role in the metastasis of various organs. Recently, we found that increased shh producing cells correlate with enhanced migration, invasiveness and increased lymphangiogenesis in Non-small-cell lung cancer (NSCLC), but the mechanism is unknown. In this study, we investigated the function of shh pathway in human NSCLC. We performed immunohistochemistry on lung tumor biopsies and the result showed the expressions of shh, Gli1, VEGF-D and LYVE1 were enhanced in lymph node metastasis specimens compared to lymph node negative lung cancer specimens. We also investigated the role of shh signaling in the invasiveness and the motility of NSCLC. We found that Shh N-terminal peptide (N-shh) enhanced cell motility and invasiveness in NSCLC, and this process was inhibited by AAD-cyclopamine (SMO inhibitor). We observed that the mRNA expressions of shh, Gli1, VEGF-D and LYVE1 were enhanced by N-shh treatment, whereas no increase of these factors was observed when KAAD-Cyclopamine (a Shh signaling inhibitor) was treated together. In addition, we confirmed that Shh-induced lymphangiogenesis and motility/invasiveness of NSCLC are also reduced by LY294002, PI-3 Kinase/Akt inhibitor meaning that shh pathway and PI-3 Kinase/Akt are strongly related. Thus, we investigated the functional relationship between Shh and Akt signaling in the metastatic progression in NSCLC. Phosphorylation of Akt was enhanced by N-Shh, but regulated by KAAD-cyclopamine or LY294002. We further analyzed the effects of shh signaling in vitro by stimulating a HMVEC with N-shh and we observed N-shh enhanced the tube formation on matrigel, but the enhancement was inhibited upon cyclopamine or LY294002 treatment. The ROS production was increased in shh-overexpressing cells. These results indicate that shh signaling pathway can contribute to ROS generation enhancing the metastatic and lymphangiogenetic potential of tumor cells. Our findings suggest that Shh-Akt signaling processes through ROS and the activation of these pathway increases the metastatic potential of non-small cell lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5320. doi:1538-7445.AM2012-5320

The Hedgehog (Hh) signaling pathway functions as an organizer in embryonic development. Recent studies have shown constitutive activation of the hedgehog pathway is important in the development of various carcinomas including breast cancer. However, it remains unclear whether this pathway is activated in human breast cancer. Herein, we investigated the role of Shh signaling pathway in tumorigenesis of estrogen treated breast cancer cells and the molecular mechanisms underlying these effects. We showed that breast cancer cell lines express all the components of Shh signaling, albeit to different extents. Moreover, in breast cancer cells, estrogen supplementation triggered Shh up-regulation. This estrogen treated Shh expression activated the Hh pathway in a ligand-dependent manner, and increased cell proliferation. Cell proliferation was done MTT assay with various concentrations of KAAD-Cyclopamine for 24, 48, or 72 h. As a result, KAAD-Cyclopamine had anti-proliferative effect on estrogen treated breast cancer cells. Also, to determine whether the cytotoxicity induced by KAAD-Cyclopamine was due to apoptosis in estrogen-induced breast cancer cells, we measured cell cycle and the fraction of early apoptosis [Annexin V(+)/PI(-)]. Cell cycle analysis by FACS indicated that Cyclopamine treated cell lines showed an increase in the proportion of cells in sub-G1 phase, compared to untreated cells. Exposure of gastric cancer cells to Cyclopamine also resulted in the increase in the percentage of annexin V- positive and PI-negative cells. These observations demonstrate that KAAD-Cyclopamine can inhibit estrogen-induced breast cancer cell cycle progression and promote cell apoptosis. Furthermore, Western blotting analysis was used to investigate the effect of KAAD-Cyclopamine (a Shh signaling inhibitor) on estroen treated breast cancer cells. As a result, expression of cleaved PARP, caspase-3, and -9, Bax was increased. In addition, phosphorylation of Akt and of glycogensynthase kinase 3β (GSK-3β) was also reduced by treatment with KAAD-Cyclopamine. These results, taken together, suggest that Blockade of the Shh pathway by KAAD-Cyclopamine promoted apoptosis in estrogen treated breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2025. doi:1538-7445.AM2012-2025

Introduction: Sonic hedgehog homolog (Shh) is known to play a critical role in normal cellular expansion and in the patterning of the early embryo of vertebrates and invertebrates, and is also implicated in some human cancers. While the sonic hedgehog homolog (Shh) pathway has been implicated in hematopoiesis and in maintenance of hematological malignancy, there is little evidence to demonstrate its importance to differentiation therapy. Procedures: We characterized the changes in HL-60 [an acute myelogenous leukemia (AML) cell line] after blocking the Shh pathway by an antagonist of Smoothened, cyclopamine. Findings: The existence of Shh signaling pathway in HL-60 cells was demonstrated by reverse transcription and PCR. Cyclopamine at 20 μM significantly down regulated the mRNA expression of Smo, Gli1 and Gli2. The flow cytometry showed that cyclopamine mediated apoptotic death of HL-60 cells in a dose- and time-dependent manner. After 12 h and 24 h, cyclopamine at 5 μM and 10 μM significantly increased the G0/G1-phase fraction of cells. Furthermore, treatment with cyclopamine increased the expression of monocytic cell markers CD 11b and CD 14, but the expression of CD 13, CD 33 and CD 38 was unchanged in HL-60 cells. The ability of cyclopamine to induce the monocytic differentiation of HL-60 cells was further evidenced by increased Egr-1 expression in a dose-dependent manner. Cyclopamine also down-regulated the phosphorylation of Akt and ERK, but activated AMPK signaling. We also analyzed the effect of cyclopamine on the primary leukemia cells from patients with AML. Conclusions: Blocking the Shh pathway via cyclopamine causes a time- and dose-dependent apoptotic cell death, increases the proportion of cells in G0/G1-phase and induces monocytic differentiation in HL-60 cells. Cyclopamine also down-regulates the mRNA expression of genes in Shh pathways. Further investigations are needed to determine the clinical application of modulating the Shh pathway in the treatment of hematological malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 158. doi:1538-7445.AM2012-158

Backgrounds: Sonic hedgehog (Shh) signaling is known to relate to cancer progression but the detail about how to correlate and link with another signaling is debating. The purpose of this study is to investigate the link between Shh signaling, androgens and epithelial-mesenchymal transition (EMT) in prostate tumor-stroma interaction during prostate cancer (PCa) progression. Materials and Methods: We investigated cell proliferation in LNCaP PCa cells, Normal Prostate Fibroblasts (NPF), and Cancer-associated Prostate Fibroblasts (CPF) in the presence of representative androgen, dihydrotestosterone (DHT) and/or Shh signaling inhibitor, cyclopamine (Cyc). With these stimulators or inhibitors, real time RT-PCR was performed to investigate downstream transcriptional factors of Shh signaling, Gli-1, EMT markers (E-cadherin, N-cadherin, and Vimentin), and Osteonectin (ON), a possible Shh target gene shown in our previous work. Expression levels of AR, Gli-1, Ki-67, Shh, and ON in specimens from PCa patients were measured by immunohistochemical (IHC) staining and correlated with clinical data. Results: While significant cell proliferation was induced by DHT in LNCaP, NPF, and CPF, Cyc inhibited these enhanced growths. CPF tended to react to DHT more but less to Cyc than LNCaP or NPF. RT-PCR results showed Shh induced EMT and DHT induced the expression of Gli-1, ON and EMT, and those inductions were blocked by Cyc. IHC staining showed that Stromal ON (ONstr) had significant correlation with serum PSA (p=0.031), high Gleason Scores had significant correlation with Ki-67, and PSA recurrence was related significantly to Gli-1 expression and lower expression of ONstr, suggesting PCa recurrence had a significant correlation with Shh-Gli-1 signaling. Conclusions: Shh signaling is involved in tumor-stromal interaction through a link with Androgens and EMT, and thus plays a pivotal role in PCa progression. Cyclopamine may therefore be a possible therapeutic agent for blocking PCa progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1407. doi:1538-7445.AM2012-1407

Objective: Recent studies have implicated hedgehog signaling in the formation and continued growth of a variety of malignancies, including ovarian cancer. Several inhibitors of the hedgehog pathway have been identified that block the activity of the Smoothened (Smo) receptor. The goal of this study was to determine the in vitro and in vivo effects of Smo antagonists alone and in combination with chemotherapy in ovarian cancer. Methods: Expression of hedgehog signaling components (Smo, Gli1 and Gli2) was assessed in 3 pairs of parental and chemotherapy-resistant ovarian cancer cell lines (A2780ip2/A2780cp20, SKOV3ip1/SKOV3TRip2, HeyA8/HeyA8MDR) using Western blot and qPCR. Cell lines were exposed to increasing concentrations of two different Smo antagonists (Cyclopamine, LDE225) alone and in combination with carboplatin, paclitaxel, adriamycin, and topotecan. Selective knockdown of Smo, Gli1 and Gli2 was achieved using siRNA constructs. Cell viability was assessed by MTT assay and PARP cleavage was used as an indicator of apoptosis. SKOV3TRip2 orthotopic xenografts were treated with vehicle, LDE225, paclitaxel or combination therapy for 5 weeks. Tumor weight for each treatment group was measured and compared using student's t-test. Results: Expression of Smo and Gli1 was high in A2780ip2/A2780cp20, moderate in SKOV3ip2/SKOV3TRip2 and low/absent in HeyA8/HeyA8MDR. Gli2 was high in SKOV3ip2/SKOV3TRip2, moderate in A2780ip2/A2780cp20 and low in HeyA8/HeyA8MDR. Response to cyclopamine and LDE225 varied among the cell lines examined with IC50s ranging from 7.5 to >20 µM. Both agents sensitized chemotherapy-resistant cell lines to paclitaxel (5- to 26-fold, including Smo[low]/Gli1[neg] HeyA8MDR), but not to carboplatin, adriamycin, or topotecan. Selective knockdown of Gli1 and Gli2 resulted in taxane sensitization only in Gli1/2-high A2780cp20 cells (2- to 8-fold). A decrease in acetyl-α-tubulin confirmed microtubule-specific effects of Smo targeting, supporting the taxane specificity of this effect. In vivo, SKOV3TRip2 xenografts treated with LDE225 or paclitaxel alone had slightly less tumor burden than the control group (reduced by 28.1%, p=0.42 and 32.0%, p=0.40, respectively). Those treated with combined LDE225 and paclitaxel, however, had significantly less tumor burden than those treated with vehicle (70.5% reduction, p=0.015). Conclusions: Targeting the hedgehog pathway decreases cell viability and increases taxane sensitivity in taxane-resistant ovarian cancer models. Interestingly, these effects were noted even in cells with little constitutive hedgehog activity. This suggests both Gli-dependent and -independent mechanisms contribute to taxane resistance, expanding the potential use of hedgehog inhibitors to all taxane-resistant tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1728. doi:10.1158/1538-7445.AM2011-1728

[Background and Aim] TRAIL is a promising therapeutic agent for human malignancies that can activate apoptosis via mitochondria-independent (Type I) or mitochodria-dependent (Type II) death receptor signaling. However, many malignant cells are resistant to TRAIL cytotoxicity and fail to trigger Type I or Type II killing. Using cholangiocarcinoma cells, we identified a role for Hedgehog signaling via GLI transcription factors in the regulation of TRAIL cytotoxicity, and provide new mechanistic insights whereby Hedghog inhibition increases apoptosis. [Materials and Methods] The human cholangiocarcinoma cell lines KMCH, HuCCT-1, and Mz-ChA-1 were employed. Hedgehog signaling was inhibited with cyclopamine or an shRNA construct targeting smootherned (SMO). Type II TRAIL signaling was inhibited with shRNA constructs for Bid or Bim and siRNA for Bax and Bak. Antiapoptotic mediators including Inhibitor of Apoptosis Proteins (IAPs) and Bcl-2 family proteins were examined by immunoblot analysis and/or real time RT-PCR. Apoptosis was quantified morphologically by DAPI and fluorescence microscopy, and biochemically by caspase-3/7 activity. GLI transcription factor binding to the XIAP promoter was examined by EMSA and chromatin immuneprecipitation (ChIP) assay. [Results] Pharmacologic or genetic inhibition of Hedgehog signaling down-regulates both XIAP and cIAP-1 mRNA and protein expression. Knockdown of XIAP, but not cIAP-1, by small-interfering RNA markedly sensitized cells to TRAIL-induced apoptosis, suggesting XIAP is the functional target. Putative GLI-binding sites were identified in the XIAP promoter, and EMSA and ChIP assays demonstrated direct binding by GLI proteins. Sensitization to TRAIL-induced apoptosis was not altered by knockdown of the BH3-only proteins Bim and Bid, or by knockdown of Bax plus Bak, implicating a mitochondria-independent Type I pathway of apoptosis. [Conclusion] These data provide evidence for up-regulation of XIAP by an oncogenic Hedgehog pathway and demonstrate that Hedgehog inhibition sensitizes cells to TRAIL via decreasing XIAP levels. Hedgehog inhibition thereby promotes Type I death receptor signaling and sensitizes cells to apoptosis, which suggests Hedgehog and XIAP inhibition may be a therapeutic approach for TRAIL-resistant neoplasms. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4105. doi:10.1158/1538-7445.AM2011-4105