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Nanoparticle delivery systems have been shown to improve the therapeutic efficacy of anti-cancer drugs, including a variety of drugs for the treatment of hepatocellular carcinoma (HCC). However, the current systems show some limitations, and the delivery of more effective nanoparticle systems for anti-HCC drugs with better targeting ability are needed. Here, we created paclitaxel (PTX)/norcantharidin (NCTD)-loaded core–shell lipid nanoparticles modified with a tumor neovasculature-targeted peptide (Ala-Pro-Arg-Pro-Gly, APRPG) and investigated their anti-tumor effects in HCC. Core–shell-type lipid nanoparticles (PTX/NCTD-APRPG-NPs) were established by combining poly(lactic-co-glycolic acid) (PLGA)-wrapped PTX with phospholipid-wrapped NCTD, followed by modification with APRPG. For comparison, PTX-loaded PLGA nanoparticles (PTX-NPs) and PTX/NCTD-loaded core–shell-type nanoparticles without APRPG (PTX/NCTD-NPs) were prepared. The in vitro and in vivo anti-tumor effects were examined in HepG2 cells and tumor-bearing mice, respectively. Morphological and release characterization showed that PTX/NCTD-APRPG-NPs were prepared successfully and achieved up to 90% release of PTX in a sustained manner. Compared with PTX/NCTD-NPs, PTX/NCTD-APRPG-NPs significantly enhanced the uptake of PTX. Notably, the inhibition of proliferation and migration of hepatoma cells was significantly higher in the PTX/NCTD-APRPG-NP group than those in the PTX-NP and PTX/NCTD-NP groups, which reflected significantly greater anti-tumor properties as well. Furthermore, key molecules in cell proliferation and apoptosis signaling pathways were altered most in the PTX/NCTD-APRPG-NP group, compared with the PTX-NP and PTX/NCTD-NP groups. Collectively, PTX/NCTD-loaded core–shell lipid nanoparticles modified with APRPG enhance the effectiveness of anti-HCC drugs and may be an effective system for the delivery of anti-HCC drugs.

Using a panel of cancer cell lines, we characterized a novel degrader of AKT, MS21. In mutant PI3K–PTEN pathway cell lines, AKT degradation was superior to AKT kinase inhibition for reducing cell growth and sustaining lower signaling over many days. AKT degradation, but not kinase inhibition, profoundly lowered Aurora kinase B (AURKB) protein, which is known to be essential for cell division, and induced G2–M arrest and hyperploidy. PI3K activated AKT phosphorylation of AURKB on threonine 73, which protected it from proteasome degradation. A mutant of AURKB (T73E) that mimics phosphorylation and blocks degradation rescued cells from growth inhibition. Degrader-resistant lines were associated with low AKT phosphorylation, wild-type PI3K/PTEN status, and mutation of KRAS/BRAF. Pan-cancer analysis identified that 19% of cases have PI3K–PTEN pathway mutation without RAS pathway mutation, suggesting that these patients with cancer could benefit from AKT degrader therapy that leads to loss of AURKB. Significance: MS21 depletes cells of phosphorylated AKT (pAKT) and a newly identified AKT substrate, AURKB, to inhibit tumor growth in mice. MS21 is superior to prior agents that target PI3K and AKT due to its ability to selectively target active, pAKT and sustain repression of signaling to deplete AURKB. This article is highlighted in the In This Issue feature, p. 2945

Inflammatory Breast Cancer (IBC) is a highly aggressive malignancy with distinct clinical and histopathological features whose molecular basis is unresolved. Here we describe a human IBC cell line, A3250, that recapitulates key IBC features in a mouse xenograft model, including skin erythema, diffuse tumor growth, dermal lymphatic invasion, and extensive metastases. A3250 cells express very high levels of the CCL2 chemokine and induce tumors enriched in macrophages. CCL2 knockdown leads to a striking reduction in macrophage densities, tumor proliferation, skin erythema, and metastasis. These results establish IBC-derived CCL2 as a key factor driving macrophage expansion, and indirectly tumor growth, with transcriptomic analysis demonstrating the activation of multiple inflammatory pathways. Finally, primary human IBCs exhibit macrophage infiltration and an enriched macrophage RNA signature. Thus, this human IBC model provides insight into the distinctive biology of IBC, and highlights potential therapeutic approaches to this deadly disease., Inflammatory breast cancer (IBC) is an aggressive form of breast cancer with a poor prognosis. Here the authors report the characterization of a human IBC cell line recapitulating the clinical and histopathological features of the human disease, and implicating its high level of CCL2 in macrophage infiltration and tumor progression.

Purpose: The Cancer Immune Monitoring and Analysis Centers – Cancer Immunologic Data Commons (CIMAC-CIDC) network supported by the NCI Cancer Moonshot initiative was established to provide correlative analyses for clinical trials in cancer immunotherapy, using state-of-the-art technology. Fundamental to this initiative is implementation of multiplex IHC assays to define the composition and distribution of immune infiltrates within tumors in the context of their potential role as biomarkers. A critical unanswered question involves the relative fidelity of such assays to reliably quantify tumor-associated immune cells across different platforms. Experimental Design: Three CIMAC sites compared across their laboratories: (i) image analysis algorithms, (ii) image acquisition platforms, (iii) multiplex staining protocols. Two distinct high-dimensional approaches were employed: multiplexed IHC consecutive staining on single slide (MICSSS) and multiplexed immunofluorescence (mIF). To eliminate variables potentially impacting assay performance, we completed a multistep harmonization process, first comparing assay performance using independent protocols followed by the integration of laboratory-specific protocols and finally, validating this harmonized approach in an independent set of tissues. Results: Data generated at the final validation step showed an intersite Spearman correlation coefficient (r) of ≥0.85 for each marker within and across tissue types, with an overall low average coefficient of variation ≤0.1. Conclusions: Our results support interchangeability of protocols and platforms to deliver robust, and comparable data using similar tissue specimens and confirm that CIMAC-CIDC analyses may therefore be used with confidence for statistical associations with clinical outcomes largely independent of site, antibody selection, protocol, and platform across different sites.

Tumors exhibit widespread enhancer landscape reprogramming compared to normal tissue. The etiology is believed to be largely cell-intrinsic in non-hormonal cancers, attributed to such genomic alterations as focal amplification of non-coding regions, aberrant activation of transcription factors, and non-coding mutations creating de novo transcription factor binding sites. Here, using freshly resected primary CRC tumors and patient-matched adjacent normal colon epithelia, we find divergent epigenetic landscapes between primary CRC tumors and CRC cell lines. We identify a unique super-enhancer signature largely absent in cell culture. Intriguingly, this phenomenon extends to highly recurrent aberrant super-enhancers gained in CRC over patient-matched normal epithelium suggesting novel insight into the etiology of enhancer reprogramming in CRC and its downstream relevance to tumor biology. We find one such super-enhancer activated in epithelial cancer cells due to surrounding inflammation in the tumor microenvironment. We restore this super-enhancer and its expressed gene, PDZK1IP1, following treatment with cytokines or xenotransplantation into nude mice, thus demonstrating its etiology via local tumor microenvironment acquisition. Building on its known role in glucose uptake via SGLT receptors, we demonstrate mechanistically that PDZK1IP1 enhances the reductive capacity CRC cancer cells via the pentose phosphate pathway using polar metabolomic profiling. We show this activation enables efficient growth under oxidative conditions both in vitro and in vivo, challenging the previous notion that PDZK1IP1 acts as a tumor suppressor in CRC. Collectively, these observations highlight the biologic significance of epigenomic profiling on patient-matched primary specimens and identify this microenvironment-acquired super-enhancer as an oncogenic driver in the setting of the inflamed tumor.

PI3K/AKT/mTOR signaling pathway is one of the most frequently dysregulated pathway in cancer development and the serine/threonine kinase AKT functions as the key node in this pathway to regulate multiple cellular and physiological processes. AKT inhibitors that competitively bind the ATP pocket have been evaluated in clinical trials and have a tolerable toxicity profile with greater efficacy for tumors with pathway mutations; however, many PI3K/AKT pathway mutant tumors remain resistant. Proteolysis targeting chimeras (PROTACs) are a powerful targeted protein degradation technology that hijacks the cellular ubiquitin-proteasome system to induce selective polyubiquitination and degradation of the target proteins. Using this technology, we designed and developed a library of novel small-molecules putative degraders to degrade AKT by recruiting either the cereblon (CRBN) or von Hippel-Lindau (VHL) E3 ligase, that have the ability to degrade AKT in cells and lower downstream signaling to various extents. Though extensive structure-activity relationship studies on various linkers, E3 ligase ligands, and AKT binding moieties, we identified a novel VHL-recruiting AKT degrader, MS21, and characterized it using a panel of 38 cancer cell lines with diverse genotypes and tissue origins. Our results suggest that efficient pharmacologic degradation of AKT phosphorylated on threonine 308 and serine 473 leads to selective inhibition of the growth of tumor cells with alterations of HER2, PIK3CA, PTEN, or AKT1. In these PI3K/PTEN pathway mutant lines, AKT degradation by MS21 was superior to AKT kinase inhibition for reducing cell growth and sustaining lower signaling over many days, and inhibited tumor cell growths through lowering the level of Aurora Kinase B, which we found to be an AKT substrate protein. AKT degradation but not kinase inhibition profoundly lowered Aurora kinase B (AURKB) protein, which is known to be essential for cell division, and induced G2/M arrest and hyperploidy. PI3K activated AKT phosphorylation of AURKB on threonine 73, which protected it from proteasome degradation. A mutant of AURKB (T73E) that mimics phosphorylation and blocks its degradation rescued cells from growth inhibition by MS21. In addition, resistance to MS21 was found to be associated with low levels of baseline AKT phosphorylation in cells as well as mutation of either KRAS or BRAF, and resistance to MS21 could be overcome by the combination treatment of a MEK inhibitor trametinib with MS21, which not only inhibited MEK but also increased AKT phosphorylation and enhanced AKT degradation. Pan-cancer analysis identified that 19% of cases have PI3K/PTEN pathway mutation without RAS pathway mutation, suggesting that these cancer patients could benefit from AKT degrader therapy that leads to loss of AURKB. Citation Format: Jia Xu, Xufen Yu, Tiphaine C. Martin, Ankita Bansal, Kakit Cheung, Abigail Lubin, Elias Stratikopoulos, Kaitlyn M. Cahuzac, Li Wang, Ling Xie, Royce Zhou, Yudao Shen, Xuewei Wu, Shen Yao, Ruifang Qiao, Poulikos I. Poulikakos, Xian Chen, Jing Liu, Jian Jin, Ramon Parsons. AKT degradation selectively inhibits the growth of PI3K/PTEN pathway mutant cancers with wild type KRAS and BRAF by destabilizing Aurora kinase B [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB195.

INTRODUCTION: Prostatic acid phosphatase (PAP) is a soluble factor secreted by prostate luminal epithelial cells. PAP expression correlates with prostate cancer (PCa) bone metastases and poor survival. The androgenic regulation of PAP in prostate development and tumorigenesis is not fully understood. We investigated the relationship between PAP and androgens in human prostate specimens and in vivo. HYPOTHESIS AND OBJECTIVES: We hypothesized that PAP expression was independent of androgens. Our objectives were to determine the immunohistochemical expression of PAP in human fetal prostate tissue, human PCa bone metastases, and xenograft and surgical castration mouse models. METHODS: Immunohistochemical staining for PAP and three androgen-regulated proteins, the Androgen Receptor (AR), Prostate-Specific Antigen (PSA), and ETS-related gene (ERG) protein, was carried out on human fetal prostate (9.5, 11.5, 13, 16.5, 18 and 20 weeks of gestational age), archival human PCa bone metastases, and PCa mouse models. For xenograft studies, PAP-expressing PCa cell lines, LNCaP, C42B, and VCaP cells, were inoculated subcutaneously into SCID mice. A castration study with surgical or sham castration was performed after VCaP tumors were palpable. Mouse tumor growth and weight were measured biweekly, and tumor tissue isolated after mouse sacrifice. RESULTS: PAP expression was observed in the fetal prostate as early as 11.5 weeks of gestational age. Strong PAP expression was noted in all human PCa bone metastases examined, both treatment-naive and castrate-resistant (n=10). However, AR and ERG expression was absent in two of four castrate-resistant specimens. PSA was weakly expressed in human castration-resistant bone metastatic prostate specimens. In vivo, PAP expression was observed in all tumor models; however, the expression of PAP differed among androgen-sensitive models; LNCaP (low PAP), C42B (moderate PAP) and VCaP (high PAP). Castrated VCaP tumors underwent tumor stasis and were significantly smaller compared to intact mice. Strong expression of PAP was observed after castration. In contrast, AR, PSA, and ERG expression were reduced in castrated VCaP tumors compared to tumors from intact mice. Double staining of tumors for PAP and AR demonstrated a population of cells that were positive for PAP but negative for AR expression located in hypoxic areas near necrosis. CONCLUSIONS: Our findings demonstrated that PAP is expressed early in normal human fetal prostate development prior to the secretion of significant androgens or expression of AR. In mouse xenografts and human PCa bone metastases, androgens did not significantly regulate PAP expression. These data demonstrate that PAP is a marker of early progenitor cells in the normal prostate and is persistently expressed after castration. PAP may be a suitable target for the treatment of castration-resistant metastatic disease.

Introduction: Prostatic Acid Phosphatase (PAP), a protein phosphatase and 5’ecto-nucleotidase, is expressed in prostate cancer (PCa) bone metastases and correlates with poor survival. Growing evidence suggests that PAP is not regulated by androgens, but rather by factors in the tumor microenvironment. Hypothesis: We hypothesized that PAP is a marker for a more progenitor type PCa cell and its expression is androgen-independent, persisting in castration-resistant disease. Methods: Protein expression of PAP and three androgen-regulated proteins, the Androgen Receptor (AR), Prostate-Specific Antigen (PSA), and ETS-related gene (ERG) protein, was assessed with immunohistochemistry in human fetal prostate (9.5 - 20 weeks of gestational age), archival human PCa bone metastases, and human PCa cell lines. VCaP cells were treated in vitro with dihydrotestosterone (DHT) and the effects on AR and PAP protein expression determined with Western Blotting. PAP-expressing PCa cell lines (LNCaP, C42B, and VCaP) were inoculated subcutaneously (s.c.) into SCID mice. To model tumor-bone interaction, LNCaP and MC3T3 osteoblast cells were co-inoculated s.c. into SCID mice. A VCaP castration study with surgical or sham castration was performed after tumors were palpable and effects of castration on tumor growth and protein expression determined. Results: PAP expression was observed in the fetal prostate as early as 11.5 weeks of gestational age prior to PSA and AR expression. Strong PAP expression was noted in all human PCa bone metastases examined, both treatment-naive and castrate-resistant (n=10). In vitro, VCaP cells expressed high levels of AR and PAP protein and DHT treatment increased AR and decreased PAP protein expression. In vivo, PAP expression was observed in all tumor models; LNCaP (low PAP expression), C42B (moderate PAP expression) and VCaP (high PAP expression). Castrated VCaP tumors underwent tumor stasis, were significantly smaller compared to intact mice, had decreased AR, PSA and ERG expression but persistent expression of PAP. Double staining of tumors for PAP and AR demonstrated a population of cells that were positive for PAP but negative for AR expression in hypoxic areas near necrosis. Inoculation of LNCaP cells with MC3T3 osteoblastic cells increased PAP expression in vivo. Conclusions: PAP is expressed early in human fetal prostate development prior to the secretion of significant androgens or expression of AR. In mouse xenograft tumors and human PCa bone metastases, androgens did not significantly regulate PAP expression. Both hypoxia and stroma increased PAP expression. These data demonstrate that PAP is a marker of early progenitor cells, is persistently expressed after castration and is upregulated by tumor microenvironmental factors. PAP may be a suitable target for the treatment of castration-resistant metastatic disease.

Targeted cancer therapies, which act on specific cancer-associated molecular targets, are predominantly inhibitors of oncogenic kinases. While these drugs have achieved some clinical success, the inactivation of kinase signaling via stimulation of endogenous phosphatases has received minimal attention as an alternative targeted approach. Here, we have demonstrated that activation of the tumor suppressor protein phosphatase 2A (PP2A), a negative regulator of multiple oncogenic signaling proteins, is a promising therapeutic approach for the treatment of cancers. Our group previously developed a series of orally bioavailable small molecule activators of PP2A, termed SMAPs. We now report that SMAP treatment inhibited the growth of KRAS-mutant lung cancers in mouse xenografts and transgenic models. Mechanistically, we found that SMAPs act by binding to the PP2A Aα scaffold subunit to drive conformational changes in PP2A. These results show that PP2A can be activated in cancer cells to inhibit proliferation. Our strategy of reactivating endogenous PP2A may be applicable to the treatment of other diseases and represents an advancement toward the development of small molecule activators of tumor suppressor proteins.

Non-small cell lung cancer (NSCLC) is one of the most fatal types of cancer with significant mortality and morbidity worldwide. MicroRNAs (miRs) have been confirmed to have positive functions in NSCLC. In the present study, we try to explore the role of miR-758 in proliferation, migration, invasion, and apoptosis of NSCLC cells by regulating high-mobility group box (HMGB) 3 (HMGB3.) NSCLC and adjacent tissues were collected. Reverse transcription quantitative PCR (RT-qPCR) was employed to detect expression of miR-758 and HMGB3 in NSCLC and adjacent tissues, in BEAS-2B cells and NSCLC cell lines. The targetted relationship between miR-758 and HMGB3 was identified by dual luciferase reporter gene assay. The effects of miR-758 on proliferation, migration, invasion, cell cycle, and apoptosis of A549 cells. MiR-758 expression was lower in NSCLC tissues, which was opposite to HMGB3 expression. The results also demonstrated that miR-758 can target HMGB3. The cells transfected with miR-758 mimic had decreased HMGB3 expression, proliferation, migration, and invasion, with more arrested cells in G1 phase and increased apoptosis. Our results supported that the overexpression of miR-758 inhibits proliferation, migration, and invasion, and promotes apoptosis of NSCLC cells by negative regulating HMGB2. The present study may provide a novel target for NSCLC treatment.

SummaryThe H3K9me3 repressive histone conformation of p53 target promoters is abrogated in response to p53 activation by MDM2-mediated SUV39H1 degradation. Here, we present evidence that the USP7 deubiquitinase protects SUV39H1 from MDM2-mediated ubiquitination in the absence of p53 stimulus. USP7 occupies p53 target promoters in unstressed conditions, a process that is abrogated with p53 activation associated with loss of the H3K9me3 mark on these same promoters. Mechanistically, USP7 forms a trimeric complex with MDM2 and SUV39H1, independent of DNA, and modulates MDM2-dependent SUV39H1 ubiquitination. Furthermore, we show that this protective function of USP7 on SUV39H1 is independent of p53. Finally, USP7 blocking cooperates with p53 in inducing apoptosis by enhancing p53 promoter occupancy and dependent transactivation of target genes. These results uncover a layer of the p53 transcriptional program mediated by USP7, which restrains relaxation of local chromatin conformation at p53 target promoters.

MicroRNAs (miRNAs/miRs) are frequently differentially expressed in non-small cell lung cancer (NSCLC), and differential miRNAs expression may be closely associated with NSCLC genesis and development. Therefore, an in-depth investigation of the cancer-associated miRNAs that are crucial for NSCLC pathogenesis may provide effective therapeutic targets for patients with this aggressive malignant tumor type. The expression levels and roles of miR-877 have been well studied in hepatocellular carcinoma and renal cell carcinoma. However, the expression pattern and functions of miR-877 in NSCLC as well as associated underlying mechanisms, to the best of our knowledge, have not yet been investigated. The present study revealed that miR-877 expression was downregulated in NSCLC tissues and cell lines. Low miR-877 expression was significantly associated with TNM stage and distant metastasis in patients with NSCLC. Functional experiments demonstrated that recovery of miR-877 expression restricted the proliferation and invasion of NSCLC cells. In addition, bioinformatics analysis predicted insulin-like growth factor 1 receptor (IGF-1R) as a potential target of miR-877. Luciferase reporter assays, reverse transcription-quantitative PCR and western blot analysis further validated that IGF-1R was a direct target of miR-877 in NSCLC. Furthermore, IGF-1R expression was markedly upregulated in NSCLC tissues, and exhibited an inverse correlation with miR-877 expression. Additionally, IGF-1R overexpression reversed the inhibitory effects in NSCLC cells caused by miR-877 upregulation. These findings demonstrated that miR-877 attenuated NSCLC cell proliferation and invasion, at least partly, by downregulating IGF-1R expression, thereby providing an new candidate biomarker for the diagnosis and therapy of patients with NSCLC.

Primary prostate cancer is generally treatable by androgen deprivation therapy, however, later recurrences of castrate-resistant prostate cancer (CRPC) that are more difficult to treat nearly always occur due to aberrant reactivation of the androgen receptor (AR). In this study, we report that CRPC cells are particularly sensitive to the growth-inhibitory effects of reengineered tricyclic sulfonamides, a class of molecules that activate the protein phosphatase PP2A, which inhibits multiple oncogenic signaling pathways. Treatment of CRPC cells with small-molecule activators of PP2A (SMAP) in vitro decreased cellular viability and clonogenicity and induced apoptosis. SMAP treatment also induced an array of significant changes in the phosphoproteome, including most notably dephosphorylation of full-length and truncated isoforms of the AR and downregulation of its regulatory kinases in a dose-dependent and time-dependent manner. In murine xenograft models of human CRPC, the potent compound SMAP-2 exhibited efficacy comparable with enzalutamide in inhibiting tumor formation. Overall, our results provide a preclinical proof of concept for the efficacy of SMAP in AR degradation and CRPC treatment. Significance: A novel class of small-molecule activators of the tumor suppressor PP2A, a serine/threonine phosphatase that inhibits many oncogenic signaling pathways, is shown to deregulate the phosphoproteome and to destabilize the androgen receptor in advanced prostate cancer. Cancer Res; 78(8); 2065–80. ©2018 AACR.

SummaryWnt canonical signaling is critical for normal development as well as homeostasis of several epithelial tissues, and constitutive activation of this pathway is commonly observed in carcinomas. We show here that 50% of human sarcomas (n = 45) and 65% of sarcoma cell lines (n = 23) of diverse histological subtypes exhibit upregulated autocrine canonical Wnt signaling. Furthermore, in Wnt autocrine cell lines, we identify alterations including overexpression or gene amplification of Wnt ligands and/or LRP5/6 coreceptors and epigenetic silencing of different cell surface Wnt antagonists. Mutations in adenomatous polyposis coli (APC) gene were observed in two nonautocrine Wnt-positive sarcoma cell lines. Finally, downregulation of the activated Wnt pathway inhibited sarcoma cell proliferation both in vitro and in vivo by a mechanism involving the downregulation of CDC25A.

Epidemiologic evidence suggests that a diet rich in fruits and vegetables is associated with a reduced risk of prostate cancer (PCa) development. Although several dietary compounds have been tested in preclinical PCa prevention models, no agents have been identified that either prevent the progression of premalignant lesions or treat advanced disease. Momordica charantia, known as bitter melon in English, is a plant that grows in tropical areas worldwide and is both eaten as a vegetable and used for medicinal purposes. We have isolated a protein, designated as MCP30, from bitter melon seeds. The purified fraction was verified by SDS-PAGE and mass spectrometry to contain only 2 highly related single chain Type I ribosome-inactivating proteins (RIPs), alpha-momorcharin and beta-momorcharin. MCP30 induces apoptosis in PIN and PCa cell lines in vitro and suppresses PC-3 growth in vivo with no effect on normal prostate cells. Mechanistically, MCP30 inhibits histone deacetylase-1 (HDAC-1) activity and promotes histone-3 and -4 protein acetylation. Treatment with MCP30 induces PTEN expression in a prostatic intraepithelial neoplasia (PIN) and PCa cell lines resulting in inhibition of Akt phosphorylation. In addition, MCP30 inhibits Wnt signaling activity through reduction of nuclear accumulation of beta-catenin and decreased levels of c-Myc and Cyclin-D1. Our data indicate that MCP30 selectively induces PIN and PCa apoptosis and inhibits HDAC-1 activity. These results suggest that Type I RIPs derived from plants are HDAC inhibitors that can be utilized in the prevention and treatment of prostate cancer.

Angiogenesis inhibition has been shown to enhance the therapeutic efficacy of cytotoxic chemotherapy in colorectal cancer. The basis of the contribution of this modality has not been defined fully. To determine the potential role of hypoxia-induced apoptosis, we studied a series of colon cancer cell lines with varying susceptibility to hypoxia. We exposed HT29 and HCT116 colon adenocarcinoma cell lines to sublethal periods of hypoxia three times weekly for 40 exposures, and derived cell lines both more resistant (from HT29) and more sensitive (from HCT116) to hypoxia-induced apoptosis. Both hypoxia-derived cell lines demonstrated more rapid growth than the parental lines when implanted subcutaneously in immunodeficient mice. Treatment of tumor-bearing mice with bevacizumab resulted in depletion of tumor microvasculature, upregulation of Hypoxia-inducible factor-1 alpha (HIF-1alpha), and increased pimonidazole staining, consistent with an anti-angiogenic effect and induction of hypoxia in tumors derived from all cell lines. The proportion of apoptotic cells was increased in all the treated tumors, and was most pronounced in the bevacizumab-treated HCT116-derived cells. The bevacizumab-treated tumors showed growth delay in HT29 and its derivative, and the parental HCT116. In the hypoxia-sensitive HCT116-derived tumors, marked tumor shrinkage and prolonged growth control occurred. Therefore, bevacizumab treatment is an effective inducer of a hypoxic environment, but the resulting cell kill and tumor shrinkage is determined by the susceptibility of the tumor to apoptosis. The induction of apoptosis by hypoxia may contribute to the benefits of such treatment in the clinical setting.

Kruppel-like factor (KLF) 6 is a tumor-suppressor gene functionally inactivated by loss of heterozygosity, somatic mutation and/or alternative splicing that generates a dominant-negative splice form, KLF6-SV1. Wild-type KLF6 (wtKLF6) expression is decreased in many human malignancies, which correlates with reduced patient survival. Additionally, loss of the KLF6 locus in the absence of somatic mutation in the remaining allele occurs in a number of human cancers, raising the possibility that haploinsufficiency of the KLF6 gene alone contributes to cellular growth dysregulation and tumorigenesis. Our earlier studies identified the cyclin-dependent kinase inhibitor p21 as a transcriptional target of the KLF6 gene in cultured cells, but not in vivo. To address this issue, we have generated two genetic mouse models to define the in vivo role of KLF6 in regulating cell proliferation and p21 expression. Transgenic overexpression of KLF6 in the liver resulted in a runted phenotype with decreased body and liver size, with evidence of decreased hepatocyte proliferation, increased p21 and reduced proliferating cell nuclear antigen expression. In contrast, mice with targeted deletion of one KLF6 allele (KLF6+/-) display increased liver mass with reduced p21 expression, compared to wild type littermates. Moreover, in primary hepatocellular carcinoma samples, there is a significant correlation between wtKLF6 and p21 mRNA expression. Combined, these data suggest that haploinsufficiency of the KLF6 gene may regulate cellular proliferation in vivo through decreased transcriptional activation of the cyclin-dependent kinase inhibitor p21.

Purpose: We investigated the role of the KLF6 tumor suppressor gene and its alternatively spliced isoform KLF6-SV1 in epithelial ovarian cancer (EOC).Experimental Design: We first analyzed tumors from 68 females with EOC for KLF6 gene inactivation using fluorescent loss of heterozygosity (LOH) analysis and direct DNA sequencing and then defined changes in KLF6 and KLF6-SV1 expression levels by quantitative real-time PCR. We then directly tested the effect of KLF6 and KLF6-SV1 inhibition in SKOV-3 stable cell lines on cellular invasion and proliferation in culture and tumor growth, i.p. dissemination, ascites production, and angiogenesis in vivo using BALB/c nu/nu mice. All statistical tests were two sided.Results: LOH was present in 59% of samples in a cell type–specific manner, highest in serous (72%) and endometrioid (75%) subtypes, but absent in clear cell tumors. LOH was significantly correlated with tumor stage and grade. In addition, KLF6 expression was decreased in tumors when compared with ovarian surface epithelial cells. In contrast, KLF6-SV1 expression was increased ∼5-fold and was associated with increased tumor grade regardless of LOH status. Consistent with these findings, KLF6 silencing increased cellular and tumor growth, angiogenesis, and vascular endothelial growth factor expression, i.p. dissemination, and ascites production. Conversely, KLF6-SV1 down-regulation decreased cell proliferation and invasion and completely suppressed in vivo tumor formation.Conclusion: Our results show that KLF6 and KLF6-SV1 are associated with key clinical features of EOC and suggest that their therapeutic targeting may alter ovarian cancer growth, progression, and dissemination.

Hypoxia is an important selective force in the clonal evolution of tumours. Through HIF-1 and other transcription factors combined with tumour-specific genetic alterations, hypoxia is a dominant factor in the angiogenic phenotype. Cellular adaptation to hypoxia is an important requirement of tumour progression independent of angiogenesis. The adaptive changes, insofar as they alter hypoxia-induced apoptosis, are likely to determine responsiveness to antiangiogenic strategies. To investigate this adaptation of tumour cells to hypoxia, we recreated in vitro the in vivo situation of chronic intermittent exposure to low-oxygen levels. The colon carcinoma cell lines HT29 and HCT116 were subjected to 40 episodes of sublethal hypoxia ( 4 h) three times a week. The resulting two hypoxia-conditioned cell lines have been maintained in culture for more than 2 years. In both cell lines changes in doubling times occurred: in HT29 an increase, and in HCT116 a decrease. Cell survival in response to hypoxia and to DNA damage differed strikingly in the two cell lines. The HT29 hypoxia-conditioned cells were more resistant than the parental line to a 24 h hypoxic challenge, while those from HCT116 surprisingly were more sensitive. Sensitivity to cisplatin in vitro was also significantly different for the hypoxia-conditioned compared with the parental lines, suggesting a change in pathways leading to apoptosis following DNA damage signaling. The growth of both conditioned cell lines in vivo as xenografts in immunodeficient ( SCID) mice was more rapid than their parental lines, and was accompanied in each by evidence of enhanced vascular proliferation as a consequence of the hypoxia-conditioning. Thus the changes in apoptotic susceptibility were independent of altered angiogenesis. The derivation of these lines provides a model for events within hypoxic regions of colon cancers, and for the acquisition of resistance and sensitivity characteristics that may have therapeutic implications for the use of antiangiogenesis drugs.

The majority of human prostate cancers arise from the peripheral zone (PZ). Prostate epithelial stem cells have been localized to the basal epithelial cell compartment. In addition, basal cells have been shown to maintain luminal epithelial cell differentiation and may mediate signals between the stromal and luminal cell compartments. Therefore, the study of adult prostate basal cells derived from different prostate zones may give insights into the mechanisms underlying normal and abnormal prostate growth. We herein compare the basal and sex steroid-stimulated expression and activity of several genes/proteins that are known to be critical in prostate cancer development in primary cultures of basal cells derived from the transition zone (TZ) and PZ of prostatectomy specimens. Our results demonstrate that prostate basal cells derived from the PZ versus TZ are more viable in culture, particularly in response to sex steroid addition. PZ cells exhibit higher telomerase activity and increased expression levels of androgen receptor, the anti-apoptotic protein bcl-2, and the dominant-negative splice variant of Kruppel-like Factor 6. PZ cells have lower basal expression levels of estrogen receptor-beta, the pro-apoptotic protein Bax, and cell-cycle inhibitor proteins (p53, p21(waf1/Cip1)). Finally, we demonstrate divergent responses to sex hormones in the two basal cell populations. The gene expression pattern in the PZ cells may partially explain the predominance of prostate cancer development in this region.

Hypoxia-induced up-regulation of vascular endothelial growth factor (VEGF) expression is a critical event leading to tumor neovascularization. Hypoxia stimulates hypoxia-inducible factor-1alpha (HIF-1alpha), a transcriptional activator of VEGF. Cyclooxygenase (COX)-2, an inducible enzyme that catalyzes the formation of prostaglandins (PGs) from arachidonic acid, is also induced by hypoxia. We reported previously that COX-2 inhibition prevents hypoxic up-regulation of VEGF in human prostate cancer cells and that prostaglandin E(2) (PGE(2)) restores hypoxic effects on VEGF. We hypothesized that PGE(2) mediates hypoxic effects on VEGF by modulating HIF-1alpha expression. Addition of PGE(2) to PC-3ML human prostate cancer cells had no effect on HIF-1alpha mRNA levels. However, PGE(2) significantly increased HIF-1alpha protein levels, particularly in the nucleus. This effect of PGE(2) largely results from the promotion of HIF-1alpha translocation from the cytosol to the nucleus. PGE(2) addition to PC-3 ML cells transfected with a GFP-HIF-1alpha vector induced a time-dependent nuclear accumulation of the HIF-1alpha protein. Two selective COX-2 inhibitors, meloxicam and NS398, decreased HIF-1alpha levels and nuclear localization, under both normoxic and hypoxic conditions. Of several prostaglandins tested, only PGE(2) reversed the effects of a COX-2 inhibitor in hypoxic cells. Finally, PGE(2) effects on HIF-1alpha were specifically inhibited by PD98059 (a MAPK inhibitor). These data demonstrate that PGE(2) production via COX-2-catalyzed pathway plays a critical role in HIF-1alpha regulation by hypoxia and imply that COX-2 inhibitors can prevent hypoxic induction of HIF-mediated gene transcription in cancer cells.

Cyclooxygenase (COX)-2 expression and prostaglandin E(2) (PGE(2)) secretion are increased in prostatic intraepithelial neoplasia (PIN) and prostate cancer. PGE(2) biosynthesis by cyclooxygenase (COX)-2 plays a pivotal role in inflammation and carcinogenesis. One of the critical proinflammatory cytokines in the prostate is interleukin-6 (IL-6). We hypothesized that increased expression of COX-2, with resultant increased levels of PGE(2) in human PIN cells, activates the IL-6 signaling pathway. We demonstrate an autocrine upregulation of PGE(2) mediated by IL-6 in a human PIN cell line. We further demonstrate that PGE(2) stimulates soluble IL-6 receptor (sIL-6R) release, gp130 dimerization, Stat-3 protein phosphorylation, and DNA binding activity. These events, induced by PGE(2), lead to increased PIN cell growth. Treatment of PIN cells with a selective COX-2 inhibitor decreases cell growth. Finally, PGE(2)-stimulated PIN cell growth was abrogated by the addition of IL-6 neutralizing antibodies. These data provide mechanistic evidence that increased expression of COX-2/PGE(2) contributes to prostate cancer development and progression via activation of the IL-6 signaling pathway.

In prostate cancer (PCa), the functional synergy between androgen receptor (AR) and nuclear factor-κ B (NF-κB) escalates the resistance to therapeutic regimens and promotes aggressive tumor growth. Although the underlying mechanisms are less clear, gene regulatory abilities of coactivators can bridge the transcription functions of AR and NF-κB. The present study shows that MYST1 (MOZ, YBF2 and SAS2, and TIP60 protein 1) costimulates AR and NF-κB functions in PCa cells. We demonstrate that activation of NF-κB promotes deacetylation of MYST1 by sirtuin 1. Further, the mutually exclusive interactions of MYST1 with sirtuin 1 vs AR regulate the acetylation of lysine 16 on histone H4. Notably, in AR-lacking PC3 cells and in AR-depleted LNCaP cells, diminution of MYST1 activates the cleavage of poly(ADP-ribose) polymerase and caspase 3 that leads to apoptosis. In contrast, in AR-transformed PC3 cells (PC3-AR), depletion of MYST1 induces cyclin-dependent kinase (CDK) N1A/p21, which results in G2M arrest. Concomitantly, the levels of phospho-retinoblastoma, E2F1, CDK4, and CDK6 are reduced. Finally, the expression of tumor protein D52 (TPD52) was unequivocally affected in PC3, PC3-AR, and LNCaP cells. Taken together, the results of this study reveal that the functional interactions of MYST1 with AR and NF-κB are critical for PCa progression.

Cyclooxygenase (COX)-2, an inducible enzyme which catalyzes the formation of prostaglandins from arachidonic acid, is expressed in prostate cancer specimens and cell lines. To evaluate the in vivo efficacy of a COX-2 inhibitor in prostate cancer, NS398 was administered to mice inoculated with the PC-3 human prostate cancer cell line.A total of 28 male nude mice were inoculated subcutaneously with 1 million PC-3 cells. Tumors were palpable in all 28 animals 1 week after inoculation and mice were randomized to receive either vehicle (control) or NS398, 3 mg./kg. body weight, intraperitoneally three times weekly for 9 weeks. Tumors were measured at weekly intervals. After a 10-week experimental period, mice were euthanized and tumors were immuno- histochemically assayed for proliferation (PCNA), apoptosis (TUNEL) and microvessel density (MVD) (Factor-VIII-related antigen). Tumor VEGF content was assayed by Western blotting.NS398 induced a sustained inhibition of PC-3 tumor cell growth and a regression of existing tumors. Average tumor surface area from control mice was 285 mm.2 as compared with 22 mm.2 from treated mice (93% inhibition, p0.001). Immunohistochemical analysis revealed that NS398 had no effect on proliferation (PCNA), but induced apoptosis (TUNEL) and decreased MVD (angiogenesis). VEGF expression was also significantly down regulated in the NS398-treated tumors.These results demonstrate that a selective COX-2 inhibitor suppresses PC-3 cell tumor growth in vivo. Tumor growth suppression is achieved by a combination of direct induction of tumor cell apoptosis and down regulation of tumor VEGF with decreased angiogenesis

Upregulation of vascular endothelial growth factor (VEGF) expression induced by hypoxia is crucial event leading to neovascularization. Cyclooxygenase-2, an inducible enzyme that catalyzes the formation of prostaglandins (PGs) from arachidonic acid, has been demonstrated to be induced by hypoxia and play role in angiogenesis and metastasis. To investigate the potential effect of COX-2 on hypoxia-induced VEGF expression in prostate cancer. We examined the relationship between COX-2 expression and VEGF induction in response to cobalt chloride (CoCl2)-simulated hypoxia in three human prostate cancer cell lines with differing biological phenotypes. Northern blotting and ELISA revealed that all three tested cell lines constitutively expressed VEGF mRNA, and secreted VEGF protein to different degrees (LNCaP > PC-3 > PC3ML). However, these cell lines differed in the ability to produce VEGF in the presence of CoCl2-simulated hypoxia. CoCl2 treatment resulted in 40% and 75% increases in VEGF mRNA, and 50% and 95% in protein secretion by LNCaP and PC-3 cell lines, respectively. In contrast, PC-3ML cell line, a PC-3 subline with highly invasive, metastatic phenotype, exhibits a dramatic upregulation of VEGF, 5.6-fold in mRNA and 6.3-fold in protein secretion after treatment with CoCl2. The upregulation of VEGF in PC-3ML cells is accompanied by a persistent induction of COX-2 mRNA (6.5-fold) and protein (5-fold). Whereas COX-2 expression is only transiently induced in PC-3 cells and not affected by CoCl2 in LNCaP cells. Moreover, the increases in VEGF mRNA and protein secretion induced by CoCl2 in PC-3ML cells were significantly suppressed following exposure to NS398, a selective COX-2 inhibitor. Finally, the effect of COX-2 inhibition on CoCl2-induced VEGF production was reversed by the treatment with exogenous PGE2. Our data demonstrate that VEGF induction by cobalt chloride-simulated hypoxia is maintained by a concomitant, persistent induction of COX-2 expression and sustained elevation of PGE2 synthesis in a human metastatic prostate cancer cell line, and suggest that COX-2 activity, reflected by PGE2 production, is involved in hypoxia-induced VEGF expression, and thus, modulates prostatic tumor angiogenesis.

The aim of this study was to determine the efficacy and toxicity of topotecan administered as a 21-day continuous intravenous infusion in patients with advanced or metastatic adenocarcinoma of the pancreas. 26 previously untreated patients with advanced or metastatic pancreatic adenocarcinoma received topotecan at a dose of 0.5 mg/m 2 /day or 0.6 mg/m 2 /day as a continuous intravenous infusion for 21 days. Courses were repeated every 28 days. 26 patients were assessable for response and toxicity on an intent-to-treat basis. The initial 8 patients at a starting dose of 0.6 mg/m 2 /day experienced unacceptable myelosuppression and dose delays. The subsequent 18 patients, therefore began therapy at a dose of 0.5 mg/m 2 /day. The major toxicity of topotecan at this dose and schedule was myelosuppression, which was reversible and non-cumulative. There were no complete responses and two partial responses for a total response rate of 8% (95% confidence interval, 1–25%). Response durations were 17 and 45 weeks. Stable disease was seen in 3 patients. The median time to progression for all patients was 8 weeks and the median survival was 20 weeks. Topotecan given as a 21-day continuous intravenous infusion has a similar response rate and median survival to our previously reported study of the 5-day short infusion regimen in pancreatic carcinoma.

Androgen signaling via the androgen receptor is a key pathway that contributes to development, cell fate decisions, and differentiation, including that of myogenic progenitors. Androgens and synthetic steroids have well established anabolic actions on skeletal muscle. Wnt and Notch signaling pathways are also essential to myogenic cell fate decisions during development and tissue repair. However, the interactions among these pathways are largely unknown. Androgenic regulation of Wnt signaling has been reported. Nandrolone, an anabolic steroid, has been shown to inhibit Notch signaling and up-regulate Numb, a Notch inhibitor. To elucidate the mechanisms of interaction between nandrolone and Wnt/Notch signaling, we investigated the effects of nandrolone on Numb expression and Wnt signaling and determined the roles of Wnt signaling in nandrolone-induced Numb expression in C2C12 myoblasts. Nandrolone increased Numb mRNA and protein levels and T cell factor (Tcf) transcriptional activity via inhibition of glycogen synthase kinase 3β. Up-regulation of Numb expression by nandrolone was blocked by the Wnt inhibitors, sFRP1 and DKK1, whereas Wnt3a increased Numb mRNA and protein expression. In addition, we observed that the proximal promoter of the Numb gene had functional Tcf binding elements to which β-catenin was recruited in a manner enhanced by both nandrolone and Wnt3a. Moreover, site-directed mutagenesis indicated that the Tcf binding sites in the Numb promoter are required for the nandrolone-induced Numb transcriptional activation in this cell line. These results reveal a novel molecular mechanism underlying up-regulation of Numb transcription with a critical role for increased canonical Wnt signaling. In addition, the data identify Numb as a novel target gene of the Wnt signaling pathway by which Wnts would be able to inhibit Notch signaling.

PP2A is a phosphatase tumor suppressor that is dysregulated and deactivated in lung cancer. It is one of the most abundant cellular proteins and regulates the activity of numerous kinases Where achievable, restoration of PP2A function inhibits cancer progression, and notably, by the inhibition of the downstream effectors of the oncogenic kinases that initiate and drive cancer progression. In this study, we determined PP2A inactivation in human lung cancer with specific molecular genotypes and we ascertained the biological and functional consequences of PP2A reactivation. In assessing a lung cancer TMA, we identified that PP2A inactivation was correlated with poor survival and was significantly higher in patients with Kras mutations. In order to understand the therapeutic potential of restoration of PP2A activity in KRAS mutant lung cancer, our lab developed a series of small molecule activators of PP2A (SMAPs) through reverse engineering of tricyclic neuroleptic drugs. SMAP treatment of lung cancer cell lines resulted in an induction of apoptosis and decreased cell viability. Structural and biophysical studies have identified the site of drug binding and mechanism for PP2A activation by this small molecule series. Additionally, cell lines harboring drug-binding mutations were resistant to SMAP therapy as compared to wild type PP2A and EGFP control. Global phosphoproteomic analysis of SMAP treated KRAS lung cancer cell lines revealed ERK signaling as a commonly perturbed pathway in drug treated cell lines. Given the marked dephosphorylation of ERK upon treatment of cell lines with SMAPs, we overexpressed a constitutively active form of MEK (MEKDD) to blunt SMAP mediated ERK dephosphorylation to determine the relevance of ERK inactivation for the biological effects of SMAPs on cellular apoptosis. Overexpression of MEKDD resulted in a blunted apoptotic response to SMAP treatment. Single agent SMAP treatment of KRAS GEMM and xenograft mouse models of lung cancer resulted in tumor stasis, induction of tumor cell apoptosis and cell cycle arrest to comparable levels seen with a combination of AKT and MEK inhibitors. Western blotting and immunohistochemical analysis of the tumors demonstrated that SMAP treatment resulted in of ERK, AKT, and PP2A-Y307 dephosphorylation in vivo. Additionally, these compounds demonstrate favorable pharmacokinetics and show no overt toxicity. Furthermore, combination of SMAPs with kinase inhibitors further decreased tumor growth in vivo. Taken together, these findings point to therapeutic activation of PP2A as a novel strategy for the treatment of KRAS-mutant NSCLC. Citation Format: Jaya Sangodkar, Rita Tohme, Janna Kiselar, Sudeh Izadmehr, Divya Hoon, Sahar Mazhar, Abbey Perl, Danica Wiredja, Daniela Schlatzer, Shen Yao, David Kastrinsky, Neelesh Sharma, David Brautigan, Mark Chance, Alain Borczuk, Michael Ohlmeyer, Yiannis Ioannou, Goutham Narla. Therapeutic activation of protein phosphatase 2A for the treatment of lung cancer. [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 3865.

s: Fourth AACR International Conference on Frontiers in Basic Cancer Research; October 23-26, 2015; Philadelphia, PA Originally an adaptive immune system in prokaryotes, CRISPR (clustered regularly interspaced short palindromic repeats) has been recently engineered into a new DNA editing technology that enables to custom modify the genome of virtually any type of cells. In the last three years, CRISPR has been used for a wide array of applications, including the generation of genetically modified organisms and new cancer models, as well as for functional studies in cultured cells. However, despite the extraordinary possibilities offered by CRISPR, two major potential drawbacks need to be considered when using this technology: the possibility of off-target DNA editing and the necessity to derive individual clones containing the desired genetic modification, which implies a relatively high level of efficiency. To overcome these intrinsic limitations, we have devised CRISPR-barcoding, a new strategy in which a potentially functional modification in the sequence of a gene of interest is coupled with a series of silent point mutations, functioning as a genetic label for cell tracking. In parallel, a second barcode consisting of distinct silent mutations is inserted in the same cell population and used as a control for CRISPR off-target effects. The genomic DNA from the resulting mixture of CRISPR-modified and unmodified cells is then probed by real-time quantitative PCR using specific primers to assess the relative proportion of each barcode. Hence, by exposing the cells to a given selective condition, this approach can be used to functionally characterize the effects of different types of mutations of a particular gene of interest. As a proof-of-concept of our strategy, we repaired the mutated sequence of the tumor suppressor APC in colorectal cancer cells, and showed that APC restoration resulted in the inhibition of both Wnt signaling reporter activity and cell growth. To further illustrate the wide range of potential applications of this approach, we used CRISPR-barcoding to modify the sequence of other cancer-related genes, including TP53, ALK, EGFR and KRAS, in various tumor cells, and we assessed the effects of such alterations on cell growth, invasion and resistance to chemotherapy, both in vitro and in vivo. Thus, by preventing the limitations associated with the low efficiency and the potential off-target effects of DNA editing, our studies demonstrate that CRISPR-barcoding is a convenient and effective strategy to investigate the functional consequences of a specific genetic modification. Citation Format: Alexis Guernet, Dorthe Cartier, Sathish Kumar Mungamuri, Sahil Adriouch, Myriam Vezain, Francoise Charbonnier, Sophie Coutant, Shen Yao, Hassan Ainani, David Alexandre, Olivier Boyer, Stuart A. Aaronson, Youssef Anouar, Luca Grumolato. Functional analysis of oncogenic driver mutations in human cancer cells through CRISPR-barcoding. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr PR10.

According to a tumor progression model, low-grade ovarian serous carcinomas may evolve from serous borderline tumors or micropapillary tumors. We sought to investigate the role of and associations between BRAF mutational status, extracellular signal regulated kinase activation, and p16(INK4A) expression in various types of ovarian serous tumors. We analyzed 29 typical ovarian serous borderline tumors, 8 micropapillary tumors, 4 low-grade invasive ovarian serous carcinomas, and 24 high-grade invasive ovarian serous carcinomas for the BRAF mutational status at codon 600; in addition, expression levels of the downstream signaling protein extracellular signal regulated kinase and the p16(INK4A) tumor suppressor protein were assessed by immunohistochemistry. There was a decline in p16(INK4A) expression from serous borderline tumors to micropapillary tumors with almost complete loss in low-grade invasive carcinomas. High-grade carcinomas had a variable p16(INK4A) expression pattern. We found a T1799A BRAF mutation in 12 typical serous borderline tumors (41%) and 1 micropapillary tumor (12.5%). No mutations were found in the low-grade and high-grade invasive carcinomas (0%). Among the typical borderline tumors, cases with BRAF mutations tended to have stronger p16(INK4A) expression compared with cases with wild-type BRAF. No other correlations were identified between the BRAF mutational status and expression levels of the analyzed proteins. Loss of p16(INK4A) expression may be a pathogenetic factor in the progression from serous borderline tumors to low-grade invasive carcinomas. The divergent molecular profiles support the theory that high-grade carcinomas are unrelated to serous borderline tumors or low-grade carcinomas.

Accumulating data suggested that CXCR4/SDF-1 pathway may play an important role in the metastasis of tumor. We previously demonstrated that CpG ODN could enhance the metastasis of human lung cancer cell via TLR9. Here we further evaluated the possible role of CXCR4/SDF-1 pathway in the enhanced metastasis of human lung cancer 95D cells induced by CpG ODN. Our data showed down-regulation of CXCR4 expression using siRNA against CXCR4 could significantly reduce the enhanced metastasis of 95D cells induced by CpG ODN both in vitro and in vivo. These results suggested that TLR9 agonist might promote the metastasis of human lung cancer cells via CXCR4/SDF-1 pathway.

Recent evidence has implied that disruption of a limited number of defined cellular pathways is necessary and sufficient for neoplastic conversion of a variety of normal human cell types in tissue culture. We show instead that malignancy in such models results from an iterative process of clonal selection in vitro and/or in vivo. Normal human fibroblasts underwent malignant transformation after transduction with telomerase, cyclin-dependent kinase 4, dominant-negative p53, and activated Ras or MEK. Furthermore, culture conditions favoring overgrowth resulted in clonal selection, which with added Ras or MEK oncogenes led to the emergence of tumorigenic clones. Such tumors showed variable degrees of malignancy with some even exhibiting metastasis. SV40 small t antigen (ST) has been reported to be necessary and sufficient to convert human fibroblasts with these pathway aberrations to a polyclonal tumor. However, we observed that clonal tumors emerged even with ST addition. Genomic instability was markedly increased by p53 and Rb pathway abrogation. Under the same conditions, fibroblasts with these alterations failed to induce tumors, implying that genomic instability may be necessary but not sufficient for malignant transformation. These findings indicate that the minimum number of events required for malignant transformation of human fibroblasts is greater than has been enumerated by such oncogene addition strategies and support a stochastic cancer progression model initiated by four defined cellular alterations. [Cancer Res 2008;68(5):1417–26]

KRAS is the most common recurrent oncogenomic mutations driving the growth of NSCLC and accounting for ∼25% of patients with advanced NSCLC. Patients with KRAS mutations respond poorly to current therapies. Thus, novel therapies, are critically needed, to improve the lives of patients suffering from KRAS driven lung cancers. While oncogenic kinases have proven to be successful targets for cancer treatment, the therapeutic targeting of phosphatases, the key negative regulators of these same pathways, has remained largely unexplored. Through reverse engineering of tricyclic neuroleptic drugs, we developed a first-in-class series of small molecule activators of PP2A activators (SMAPs) molecules, as represented by TRC-794 and TRC-1154, that have favorable pharmaceutical properties directly bind and activate the serine/threonine phosphatase 2A (PP2A). PP2A accounts for the majority of cellular serine/threonine phosphatase activity, and its dominant and best-defined targets are oncogenic protein kinases including ERK and AKT. In this study, we sought to determine both the association of PP2A inactivation in lung cancer with specific molecular genotypes and the biological and functional consequences of PP2A reactivation in lung cancer. We determined the PP2A activation status by immunohistochemistry for the Y307 PP2A residue, a well documented inactivating site on the phosphatase, in a large cohort of primary lung tumors and identified that KRAS G12C mutant tumors displayed coordinate overexpression of both pERK and PP2A Y307. Global phosphoproteomic analysis of TRC-794 treated KRAS lung cancer cell lines revealed ERK signaling as the only commonly perturbed pathway in drug treated cell lines which was confirmed by western blotting. Treatment of lung cancer cell lines with TRC resulted in decreased cell viability, decreased colony formation, and an increase in apoptosis. Given the marked dephosphorylation of ERK upon treatment of cell lines with TRC-1154, we overexpressed a constitutively active form of MEK (MEKDD) to blunt SMAP mediated ERK dephosphorylation to determine the relevance of ERK inactivation to the biological effects of SMAPs on cellular apoptosis. Overexpression of MEKDD resulted in blunting the apoptotic response to TRC-1154 treatment. Single agent TRC-794 or TRC-1154 treatment of KRAS GEMM and xenograft mouse models of lung cancer resulted in tumor stasis, induction of tumor cell apoptosis and cell cycle arrest to comparable levels seen with a combination of AKT and MEK inhibitors. Western blotting and immunohistochemical analysis of the tumors demonstrated that SMAP treatment resulted in of ERK, AKT, and PP2A-Y307 dephosphorylation in vivo. Additionally, these compounds demonstrate favorable pharmacokinetics and show no overt toxicity. Taken together, these findings point to therapeutic activation of PP2A as a novel strategy for the treatment of advanced KRAS-mutant NSCLC. Citation Format: Jaya Sangodkar, Sudeh Izadmehr, Sahar Mahzar, Divya Hoon, Shen Yao, David Kastrinsky, Daniela Schlatzer, Neelesh Sharma, Alain C. Borczuk, Michael Ohlmeyer, Yiannis Ioannou, Goutham Narla. Development of small molecule activators of protein phosphatase 2A for the treatment of lung 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 5329. doi:10.1158/1538-7445.AM2015-5329

The OPG/RANKL/RANK system regulates osteoclastogenesis. Both cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) and interleukin-6 (IL-6) are reported to induce osteoclast differentiation. The mechanisms underlying these signaling pathways on the OPG/RANKL/RANK system are not fully understood. We herein demonstrate that COX-2 and PGE2 stimulated osteoclastogenesis through inhibition of OPG secretion, stimulation of RANKL production by osteoblasts, and upregulation of RANK expression in osteoclasts. PGE2 also stimulated IL-6 production, and IL-6, in turn, increased PGE2 secretion, COX-2, and EP4/EP2 expression in bone cells. These findings provide evidence of interactive effect of PGE2 and IL-6 signaling pathways in osteoclastogenesis via effect on the OPG/RANKL/RANK system.

Metastatic non-small cell lung cancer (NSCLC) is the most common cause of cancer death. Cytotoxic chemotherapy has historically been the mainstay of therapy but is associated with only modest improvements in patient survival. Over the past decade, a better understanding of the pathogenesis of NSCLC, coupled with high throughput genomic technologies applied to patient tumor samples, has led to a molecular classification of NSCLC and a new generation of “precision” therapies. However, the most common recurrent oncogenomic mutation driving the growth of NSCLC, mutant KRAS, accounting for ∼25% of patients with advanced NSCLC, remains without an effective targeted therapy. Mutations in KRAS lead to downstream signaling through ERK, as well as cross talk with the PI3K-Akt pathway, the latter of which is amplified in the presence of inhibition of ERK pathway signaling alone. These findings likely explain, at least in part, why targeting ERK pathway signaling alone in NSCLC has been largely unsuccessful in the clinic, and suggest that coordinate inhibition of both ERK and Akt is necessary for optimal therapy. Approaches to inhibit both of these pathways simultaneously with co-administration of two small molecular kinase inhibitors has shown some promise, but has been limited by both “off-target” treatment-limiting side effects and suboptimal coordinate inhibition of both Akt and ERK signaling. Thus, novel therapies, are critically needed, to improve the lives of patients suffering from KRAS driven lung cancers and while oncogenic kinases have proven to be successful targets for cancer treatment, the therapeutic targeting of phosphatases, the key negative regulators of these same pathways, has remained largely unexplored. Starting with the observation that tricyclic neuroleptic drugs exert anticancer effects in xenograft models, we employed combinatorial chemistry to reverse engineer these drugs into a series of novel compounds that retain the anti-proliferative effects but are devoid of the dose-limiting effects on the central nervous system. We have demonstrated these agents exert potent anti-proliferative effects in both cell culture and in vivo lung cancer models and these effects are functionally linked with simultaneous inhibition of both PI3K-Akt and MAPK signaling. Importantly, these agents that have favorable pharmaceutic properties directly bind and activate the serine/threonine phosphatase 2A (PP2A) and we call these novel first-in-class agents Small Molecule Activators of PP2A (SMAPs). A critical role for PP2A as a tumor suppressor has previously been established, and inhibition and loss-of-function changes in PP2A occur in human lung cancers. Furthermore, protein phosphatase 2A (PP2A) accounts for the majority of cellular serine/threonine phosphatase activity, and its dominant and best defined targets are protein kinases and oncogenic proteins including ERK and AKT. Here we demonstrate for the first time the development and validation of a first-in-class orally bioavailable pharmacological agent that can directly bind and activate PP2A driving coordinate inhibition of both the MAPK and AKT effector pathways in cell culture and both xenograft and genetically engineered mouse models (GEMM) of human lung cancer. Global phosphoproteomic analysis of SMAP treated KRAS lung cancer cell lines reveals ERK signaling as the only commonly perturbed pathway in drug treated cell lines. Single agent SMAP treatment of KRAS GEMM and xenograft mouse models of lung cancer resulted in tumor stasis, induction of tumor cell apoptosis and cell cycle arrest to comparable levels seen with a combination of AKT and MEK inhibitors. Additionally, the compounds demonstrate favorable pharmacokinetics and show no overt toxicity. Taken together, these findings point to therapeutic activation of PP2A as a novel strategy for the treatment of advanced KRAS-mutant NSCLC. While research and clinical effort has largely focused on development of inhibitors of oncogenic kinases, the identification of small molecule activators of tumor suppressor proteins has remained elusive. Activation of such proteins could offer the opportunity to identify novel synergistic strategies for the treatment of a number of cancer types. Nevertheless, translation of a PP2A activation strategy into clinical medicine has required pharmaceutically tractable agents for development. Our studies represent a first step into that new territory and highlight the potential for the development of small molecule activators of other protein phosphatases and tumor suppressor proteins. Citation Format: Jaya Sangodkar, Sahar Mazhar, Danica Wiredja, Giridharan Gokulrangan, Daniela Schlatzer, David Kastrinsky, Analisa Difeo, Shen Yao, Sudeh Izadmehr, Neelesh Sharma, Yiannis Ioannou, Michael Ohlmeyer, Goutham Narla. Therapeutic targeting of oncogenic KRAS signaling using a novel small molecule agonist of the PP2A tumor suppressor gene. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A38. doi: 10.1158/1557-3125.RASONC14-A38

Hypoxia contributes to cytotoxic chemotherapy and radiation resistance and may play a role in the efficacy of antiangiogenesis cancer therapy. We have generated a series of cell lines derived from the colon adenocarcinoma models HT29 and HCT116 by exposing cells in vitro to repeated sublethal periods of profound hypoxia. These cell lines have altered sensitivity to hypoxia-induced apoptosis: those derived from HT29 are resistant, whereas those from HCT116 are more susceptible. We used cDNA selected subtractive hybridization to identify novel genes mediating sensitivity to hypoxia-induced apoptosis and isolated macrophage migration inhibitory factor (MIF) from the hypoxia-conditioned cell lines. MIF expression correlates with susceptibility of the cell lines to apoptosis. In hypoxia-resistant cells, the induction of apoptosis by hypoxia can be restored by the addition of exogenous recombinant MIF protein, suggesting that resistance may result in part from down-regulation of MIF production possibly through an autocrine loop. Inhibition of MIF using small interfering RNA in the susceptible lines conferred resistance to hypoxia-induced cell death. The relative expression of MIF in the hypoxia-conditioned cells implanted s.c. in severe combined immunodeficient mice in vivo was similar to that observed in vitro. In an analysis of 12 unrelated colon tumor cell lines, MIF expression and response to hypoxia varied widely. Cell lines in which MIF was inducible by hypoxia were more sensitive to oxaliplatin. In human colon tumor specimens analyzed by immunohistochemistry, MIF expression was similarly variable. There was no detectable expression of MIF in normal colon mucosa or adenoma but positive staining in all carcinomas tested. Taken together, these data indicate that MIF may be a determinant of hypoxia-induced apoptosis in vitro and that its variable expression in human colon cancers may indicate a functional role in vivo. We suggest that MIF expression in colorectal cancer may be a marker of susceptibility to therapies that may depend on induction of hypoxia, possibly including antiangiogenic therapy.

Prostate cancer is a leading cause of cancer death in men. Risk prognostication, treatment stratification, and the development of rational therapeutic strategies lag because the molecular mechanisms underlying the initiation and progression from primary to metastatic disease are unknown. Multiple lines of evidence now suggest that KLF6 is a key prostate cancer tumor suppressor gene including loss and/or mutation in prostate cancer tumors and cell lines and decreased KLF6 expression levels in recurrent prostate cancer samples. Most recently, we identified a common KLF6 germ line single nucleotide polymorphism that is associated with an increased relative risk of prostate cancer and the increased production of three alternatively spliced, dominant-negative KLF6 isoforms. Here we show that although wild-type KLF6 (wtKLF6) acts as a classic tumor suppressor, the single nucleotide polymorphism-increased splice isoform, KLF6 SV1, displays a markedly opposite effect on cell proliferation, colony formation, and invasion. In addition, whereas wtKLF6 knockdown increases tumor growth in nude mice >2-fold, short interfering RNA–mediated KLF6 SV1 inhibition reduces growth by ∼50% and decreases the expression of a number of growth- and angiogenesis-related proteins. Together, these findings begin to highlight a dynamic and functional antagonism between wtKLF6 and its splice variant KLF6 SV1 in tumor growth and dissemination.

PURPOSE: Raf-1 is a protein kinase that plays a broad role in oncogenic signaling and acts as a downstream effector of Ras in the mitogen-activated protein kinase pathway. The present study was designed to determine the maximum-tolerated dose (MTD), toxicity profile, pharmacokinetics, and antitumor activity of the c-raf-1 antisense oligodeoxynucleotide ISIS 5132 (CGP 69846A; ISIS Pharmaceuticals Inc, Carlsbad, CA). The effect of ISIS 5132 on c-raf-1 gene expression in peripheral-blood mononuclear cells (PBMCs) of treated patients was studied using a reverse transcriptase polymerase chain reaction assay. PATIENTS AND METHODS: Patients with refractory malignancies received ISIS 5132 as a 2-hour intravenous infusion three times weekly for 3 consecutive weeks. Pharmacokinetic sampling was performed during the first cycle in all patients; PBMCs for c-raf-1 mRNA analysis were collected at baseline and on days 3, 5, 8, and 15 of cycle 1 and on day 1 of each cycle thereafter. RESULTS: Thirty-one patients received ISIS 5132 at one of nine dose levels ranging from 0.5 mg/kg to 6.0 mg/kg. Clinical toxicities included fever and fatigue, but these were not dose limiting. A clinically defined MTD was not reached. The harmonic mean half-life of ISIS 5132 was 59.8 minutes (range, 35.5 to 107.3 minutes). The area under the concentration-time curve increased linearly with dose, and mean plasma clearance was 1.86 mL/kg/min (range, 1.21 to 2.41 mL/kg/min). Two patients experienced prolonged stable disease lasting more than 7 months, which was associated with persistent reduction in c-raf-1 expression in PBMCs. Significant decreases in c-raf-1 expression were identified at time points after the baseline value (P < .05) at doses ≥ 2.5 mg/kg. CONCLUSION: ISIS 5132 is well tolerated at doses up to 6.0 mg/kg when administered as a thrice weekly 2-hour infusion for 3 consecutive weeks. The pharmacokinetic behavior of the drug is reproducible, and suppression of target gene expression is observed in circulating PBMCs.

Background Recent evidence indicates that programmed cell death or apoptosis may be an important mechanism for the lethality of cancer chemotherapy drugs in tumor cells. Apoptosis may be induced in tumor cells by a number of physical stresses, including radiation, UV light, heat shock, and cold. In preliminary studies, we have found that exposure of cells to hypoxia rapidly induces the expression of several immediate early genes, including c-jun, jun-D, and c-fos, and of a bifunctional redox protein/endonuclease, Ref-1. Purpose Our purpose was to determine if hypoxia-induced overexpression of the endonuclease was associated with altered DNA integrity and manifestations of apoptosis. Methods We examined cultured cells for the induction of apoptosis by electrophoresis and immunofluorescence techniques and related these findings to the induction of gene expression by hypoxia. Results We found that an 8-hour exposure of human adenocarcinoma HT29 cells to hypoxia (which results in only 14% loss of viability) induced internucleosomal DNA fragmentation beginning after 8 hours of hypoxia and before reoxygenation. Immunofluorescence of hypoxia-treated cells showed that apoptotic cells were detectable initially after 4 hours of hypoxia and reached a peak (31.3% of cells) at 12 hours after reoxygenation and that by 36 hours after reoxygenation all apoptotic cells had been eliminated from the population. Hypoxia-induced apoptosis was associated with a marked induction of c-myc messenger RNA (mRNA). We have previously shown that hypoxia is associated with ref-1 mRNA induction. While expression of c-myc declined with a time course similar to that of the disappearance of apoptotic cells, that of ref-1 remained elevated. Coincident with the decline of c-myc, we observed a late increase in the expression of bcl-2 beginning 24 hours after reoxygenation. Conclusions and implications These results suggest a possible relationship between Ref-1 induction and the occurrence of apoptosis following a hypoxic exposure.

The topoisomerase I inhibitor topotecan is a potent water-soluble camptothecin derivative with activity in a wide variety of preclinical models. Topotecan exhibits schedule dependency in vivo, with the greatest activity being observed on repeated dose schedules. On the basis of the initial clinical studies that showed a short plasma half-life, we attempted to prolong drug exposure by giving topotecan as a 24-h infusion weekly. In a phase I trial, we treated 32 patients at doses ranging from 1.0 to 2.0 mg/m2. The patient population had not been heavily pretreated with chemotherapy and was of good performance status. The incidence of neutropenia, which was dose-limiting, increased sharply with relatively small increments in dose. Doses greater than 1.5 mg/m2 were associated with nadirs that developed after one to three weekly treatments. A patient with metastatic colorectal cancer had a prolonged partial response. The plasma pharmacokinetics of topotecan (lactone and open forms) was characterized in 21 patients. Mean plasma steady-state drug levels were proportional to the dose and were within the range required to exert cytotoxicity in preclinical models. Plasma elimination curves were fit to a one-compartment model, in which the harmonic mean half-life of topotecan was 3.5 h. The ratio of the lactone to the total drug concentrations was constant throughout, which suggests that for this schedule the total drug concentration may be used as a measure of active lactone exposure. This conclusion is supported by the pharmacodynamic analysis, which revealed a positive correlation of both lactone and total drug steady-state concentrations with bone marrow toxicity. The further investigation of this and other infusional schedules in phase II trials will be conducted. The steady-state concentrations of total drug will be measured in several of these trials to establish its potential role in adaptive dosing using this schedule. Such a strategy is justified by the interpatient variability in toxicity and the steep dose-response curve observed in this study. Preliminary evidence of interpatient variability in the mRNA expression of topoisomerase I in the peripheral mononuclear cells and colon mucosa is presented. Trials are under way using biological endpoints for further selection of patients in whom the use of topoisomerase inhibitors may be therapeutically beneficial.