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Tumor metastasis is a complex process. A better understanding of the mechanistic underpinnings of the metastatic cascade could uncover approaches to better prevent and target metastases. Here, we integrated transcriptomic profiling in primary esophageal squamous cell carcinoma (ESCC) and matched lymph node metastatic tissues and genome-wide CRISPR/Cas9 functional screening to identify the key drivers of cancer metastasis. Anoctamin 1 (ANO1) expression was found to be elevated in primary tumors and further increased in metastatic tissues. High expression of ANO1 was associated with poor patient survival, and ANO1 overexpression promoted cancer invasion and metastasis. Mechanistically, ANO1 inactivated the LXR pathway and increased intracellular cholesterol accumulation by interacting with JUN to repress the transcription of cholesterol hydroxylase, CYP27A1. In a paracrine manner, IL1β secreted from ANO1-expressing ESCC cancer cells activated NF-κB signaling in fibroblasts to stimulate production of the chemokine CCL1, which in turn enhanced invasion of ESCC cancer cells. Furthermore, molecular docking and in silico screening of 1,500,000 small molecules identified K786–4469 as a lead compound that targeted ANO1 and suppressed cancer metastasis in vitro and in vivo. Overall, these findings uncover a noncanonical function of ANO1 in regulating cholesterol metabolism and fibroblast function, revealing ANO1 as a potential prognostic biomarker and therapeutic target for metastatic ESCC. Significance: Metastatic cancer cells upregulate ANO1 to activate cell-intrinsic and -extrinsic mechanisms that alter cholesterol metabolism and stimulate fibroblasts, which can be targeted with ANO1 inhibitors to inhibit metastatic growth. See related commentary by Singh and Mehla, p. 1759

High blood glucose has long been established as a risk factor for tumor metastasis, yet the molecular mechanisms underlying this association have not been elucidated. Here we describe that hyperglycemia promotes tumor metastasis via increased platelet activity. Administration of glucose, but not fructose, reprogrammed the metabolism of megakaryocytes to indirectly prime platelets into a prometastatic phenotype with increased adherence to tumor cells. In megakaryocytes, a glucose metabolism-related gene array identified the mitochondrial molecular chaperone glucose-regulated protein 75 (GRP75) as a trigger for platelet activation and aggregation by stimulating the Ca2+-PKCα pathway. Genetic depletion of Glut1 in megakaryocytes blocked MYC-induced GRP75 expression. Pharmacologic blockade of platelet GRP75 compromised tumor-induced platelet activation and reduced metastasis. Moreover, in a pilot clinical study, drinking a 5% glucose solution elevated platelet GRP75 expression and activated platelets in healthy volunteers. Platelets from these volunteers promoted tumor metastasis in a platelet-adoptive transfer mouse model. Together, under hyperglycemic conditions, MYC-induced upregulation of GRP75 in megakaryocytes increases platelet activation via the Ca2+-PKCα pathway to promote cancer metastasis, providing a potential new therapeutic target for preventing metastasis. Significance: This study provides mechanistic insights into a glucose–megakaryocyte–platelet axis that promotes metastasis and proposes an antimetastatic therapeutic approach by targeting the mitochondrial protein GRP75.

CCNE1 gene amplification is associated with overexpression of Cyclin E1 protein and is an important oncogenic driver. CCNE1 amplification is common in high grade serous ovarian carcinomas (HGSOC) and is associated with platinum resistance and poor patient outcomes. Importantly, Cyclin E1 overexpression can also occur in the absence of gene amplification in ovarian cancer. Overexpression of Cyclin E1 increases CDK2 activity and accelerates entry into S phase of cell cycle, resulting in replication stress and rendering cells more dependent on DNA repair. WEE1 participates in the DNA damage response by controlling essential checkpoints of the cell cycle, preventing cells from entering mitosis and allowing for DNA repair before cell cycle progression. Thus, we hypothesize that ovarian cancers overexpressing Cyclin E1, either via CCNE1 gene amplification or through other independent mechanisms, are more sensitive to WEE1 inhibition. Here, we tested this hypothesis in a range of preclinical cancer models using azenosertib, a novel, selective, and orally bioavailable WEE1 inhibitor currently in clinical development. Ovarian cancer cell lines overexpressing Cyclin E1 protein (Cyclin E1high) were more sensitive to azenosertib than ovarian cancer cell lines with lower levels of Cyclin E1 protein (Cyclin E1low) inducing significant cytotoxic effects (GRmax < -0.5). In Cyclin E1low ovarian cancer cell lines, overexpression of Cyclin E1 increased sensitivity to azenosertib. In contrast, silencing of CDK2 in Cyclin E1high cells reduced sensitivity to azenosertib. In vivo, higher levels of baseline expression of Cyclin E1 in tumor cells were associated with better response to azenosertib, with tumor growth inhibition (TGI) ranging from 51.5% in a Cyclin E1low SKOV3 model to 88% in a Cyclin E1high OVCAR3 model. Azenosertib synergized with chemotherapy agents in multiple ovarian cancer models. In vitro, we report that Cyclin E1high cancer cells were more sensitive to the combination of azenosertib and chemotherapeutics than Cyclin E1low cancer cells. In vivo, the synergy between azenosertib and paclitaxel was stronger in Cyclin E1high OVCAR3 model (46% reduction of initial tumor volume/104% TGI) than Cyclin E1low A2780 model (85% TGI). Taken together, these data suggest that Cyclin E1 overexpression via gene amplification or independent mechanisms sensitize ovarian cancer cells to azenosertib alone or in combination with chemotherapy. This observation supports the use of Cyclin E1 expression to enrich for azenosertib responders in ovarian cancer and possibly other tumor types. Two subsets of Cyclin E1-driven platinum resistant ovarian cancer patients, as defined by gene amplification or protein overexpression, are being enrolled in an azenosertib monotherapy trial. Citation Format: Jianhui Ma, Wen Liu, Heekyung Chung, Hooman Izadi, Petrus DeJong, Olivier Harismendy, Jia li, Fernando Doñate, Ahmed Samatar, Mark Lackner, Laure Escoubet, Kevin Bunker, Daehwan Kim, Nathan Jameson, Tugba Yildiran Ozmen, Kangjin Jeong, Dong Zhang, Wen-An Pan, Gordon Mills. Cyclin E1 protein overexpression sensitizes ovarian cancer cells to ZN-c3, a novel, selective and oral bioavailable inhibitor of Wee1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2153.

Background Triple-negative breast cancer (TNBC) is more frequently diagnosed in young patients, with an incidence of 26% of this population compared to 12% overall, and is characterized by high malignancy and poor prognosis. Limited data are available that contribute to a comprehensive summarization of the prognostic factors and the determination of surgical strategy that are associated with young patients with TNBC. We aimed to determine the optimal surgical approach (breast-conserving versus mastectomy) for patients aged < 40 years with TNBC and establish a prognostic model. Methods We performed a cohort study with a median follow-up of 31 months using the Surveillance, Epidemiology, and End Results (SEER) data of young patients < 40 years diagnosed with stage I–III TNBC between 2010 and 2016. A Cox proportional hazards model was used to investigate the effects of baseline characteristics on breast cancer-specific survival (BCSS) and overall survival (OS). To ensure that differences in outcomes were not based on baseline differences in demographic and clinical characteristics, we performed Kaplan–Meier analysis before and after propensity score matching (1:1). Subgroup analyses stratified by TNM stage as well as further propensity score matching analyses were performed. A nomogram was constructed from the multivariate logistic regression to incorporate all the prognostic factors to predict the BCSS rates of patients at 3 years and 5 years. Young patients < 40 years diagnosed with stage I–III TNBC between 2006 and 2016 in Shenzhen Second People’s Hospital (SSPH) were enrolled as external validation. Results A total of 2,854 patients from SEER dataset and 250 from SSPH were included in this study. On multivariable analysis, unmarried status, lack of health insurance, advanced T stage, advanced N stage, invasive lobular carcinoma or mixed histologic type, were all significantly associated with poor BCSS and OS. Young patients with TNBC were more likely to undergo mastectomy than breast-conserving surgery. Notably, patients with T1N0M0 or T2-4N+M0 tumors who underwent breast-conserving surgery achieved longer BCSS and OS than those who underwent mastectomy; however, the type of surgery did not influence survival rates among patients with T1N+M0 or T2-4N0M0 tumors. The nomogram was constructed by the five variables and passed the calibration and validation steps (C-index: 0.774 for training cohort and 0.768 for validation cohort). The area under the receiver operating characteristic curves (AUCs) predicting the 3-year and 5-year BCSS rates were calculated (0.783 and 0.774 in training cohort; 0.786 and 0.772 in validating cohort). Conclusions A localized surgical approach may be a superior option for young patients with TNBC, especially those with T1N0M0 and T2-4N+M0 tumors. Marital status, health insurance status, T stage, N stage, and histological type were independent prognostic factors, and a nomogram established based on these variables successfully predicted the 3- and 5-year survival probabilities among these patients. Citation Format: Yi-Zi Zheng, Yan Liu, Zhen-Han Deng, Guo-Wen Liu, Ni Xie. Determining prognostic factors and optimal surgical intervention for young patients with triple-negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-03-06.

Macrophages perform key and distinct functions in maintaining tissue homeostasis by finely tuning their activation state. Within the tumor microenvironment, macrophages are reshaped to drive tumor progression. Here we report that tumor necrosis factor α-induced protein 8–like 1 (TIPE1) is highly expressed in macrophages and that depletion of TIPE1 impedes alternative activation of macrophages. TIPE1 enhanced activation of the PI3K/Akt pathway in macrophages by directly binding with and regulating the metabolism of phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3). Accordingly, inhibition of the PI3K/Akt pathway significantly attenuated the effect of TIPE1 on macrophage alternative activation. Tumor-associated macrophages (TAM) in human liver cancer and melanoma tissues showed significantly upregulated TIPE1 expression that negatively correlated with patient survival. In vitro and in vivo, TIPE1 knockdown in macrophages retarded the growth and metastasis of liver cancer and melanoma. Furthermore, blockade or depletion of TGFβ signaling in macrophages abrogated the effects of TIPE1 on tumor cell growth and migration. Together, these results highlight that the phosphoinositide-related signaling pathway is involved in reprogramming TAMs to optimize the microenvironment for cancer progression. Significance: This work provides insight into the fine tuning of macrophage polarization and identifies a potential target for macrophage-based antitumor therapy.

As a cytoplasmic sensor of double-stranded DNA (dsDNA), the cyclic GMP-AMP synthase–stimulator of IFN genes (STING) pathway plays an important role in antitumor immunity. In this study, we investigated the effect of Src homology-2 domain-containing protein tyrosine phosphatase-2 (SHP2) on tumor cell–intrinsic STING pathway activity and DNA repair in colon cancer. SHP2 interacted with and dephosphorylated PARP1 after DNA damage. PARP1 inhibition by SHP2 resulted in reduced DNA repair and accumulation of dsDNA in cells, thus promoting hyperactivation of the STING pathway. The SHP2 agonist lovastatin was able to enhance SHP2 activity and promote STING pathway activation. Moreover, lovastatin significantly enhanced the efficacy of chemotherapy in colon cancer models, in part via STING pathway-mediated antitumor immunity. These findings suggest that SHP2 exacerbates STING pathway activation by restricting PARP1-mediated DNA repair in tumor cells, providing a basis for the combined use of lovastatin and chemotherapy in the treatment of colon cancer. Significance: Dephosphorylation of PARP1 by SHP2 simultaneously suppresses DNA repair and enhances STING pathway-mediated antitumor immunity, highlighting SHP2 activation as a potential therapeutic approach in colon cancer.

In this study, we used a bioinformatic approach to identify genes whose expression is dysregulated in human prostate cancers. One of the most dramatically downregulated genes identified encodes CYP27A1, an enzyme involved in regulating cellular cholesterol homeostasis. Importantly, lower CYP27A1 transcript levels were associated with shorter disease-free survival and higher tumor grade. Loss of CYP27A1 in prostate cancer was confirmed at the protein level by immunostaining for CYP27A1 in annotated tissue microarrays. Restoration of CYP27A1 expression in cells where its gene was silenced attenuated their growth in vitro and in tumor xenografts. Studies performed in vitro revealed that treatment of prostate cancer cells with 27-hydroxycholesterol (27HC), an enzymatic product of CYP27A1, reduced cellular cholesterol content in prostate cancer cell lines by inhibiting the activation of sterol regulatory-element binding protein 2 and downregulating low-density lipoprotein receptor expression. Our findings suggest that CYP27A1 is a critical cellular cholesterol sensor in prostate cells and that dysregulation of the CYP27A1/27HC axis contributes significantly to prostate cancer pathogenesis. Cancer Res; 77(7); 1662–73. ©2017 AACR.

Despite the well known energy requirements and stresses of metastasis, the relationships between metabolism, mitochondrial genetics and metastasis are still underdeveloped. Two lines of investigation point to more intimate involvement of mtDNA than is widely appreciated. First, recent data demonstrate that the metastasis suppressor KISS1 essentially reverses the so-called Warburg Effect by regulating mitochondrial biogenesis. KISS1 re-expression results in higher pH[Ex] due to reduced lactate secretion concomitant with reduced glycolysis and a shift toward oxidative phosphorylation. KISS1-expressing cells have 30-50% more mitochondrial mass, which appears to be due to higher expression of PPARγ co-activator 1α (PGC1α), a master regulator of mitochondrial biogenesis. shRNA-mediated knockdown of KISS1 and PGC1α establish a pathway between these molecules, mitochondrial biogenesis and metastatic potential. Second, genetic crosses with a newly described MNX (mitochondrial-nuclear exchange) mice suggest that mitochondrial polymorphisms (haplotypes) may control susceptibility to metastasis. Transgenic FVB/N-tg:MMTV-PyMT which spontaneously develop mammary tumors and lung metastasis with high penetrance were crossed with female MNX mice having the same nuclear background (FVB - wild-type) but with C57BL/6 and BALB/c mitochondrial backgrounds. Using this strategy, the mtDNA contributions to metastasis can be discriminated. Results demonstrate that tumor and metastasis incidence do not appear to be significantly different. However, metastasis size is greatly affected. Taken together, these data strongly support the concept that mitochondrial-nuclear cross-talk is a more significant determinant of metastasis than generally appreciated. SUPPORT: NCI-CA134981; Natl Fndn Cancer Res, Susan G. Komen SAC11037, Hall Family Fndn, KS Bioscience Auth. Citation Format: Danny R. Welch, Wen Liu, Kyle P. Feeley, Scott W. Ballinger. Nuclear-mitochondrial cross-talk: A key determinant of cancer metastasis. [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 SY20-03. doi:10.1158/1538-7445.AM2014-SY20-03

Objective: Peripheral T-cell lymphoma (PTCL) is a type of non-hodgkin lymphoma with high metastasis and poor prognosis. The guidelines (NCCN guidelines, 2017) recommended CHOEP or CHOP chemotherapy regimen for treatment of PTCL. Unfortunately, the 5-year survival rate of PTCL is lower than 30% according to the present clinical data. Establishing new effective treatment methods and strategies for PTCL has been an urgent problem. Car-T cell therapy is a promising therapy for individuals with lymphoma. In USA, CD19 Car-T cell therapy has been proved to be effective for acute and chronic B-cell lymphoma treatment in more than 1000 clinic trials. However, there is currently no known Car-T cell therapy for PTCL, one of the most prevalent malignancies in Asia. In this study, to provide a safe and effective treatment for the recurrence and malignant CD30 positive PTCL, we establish the world-first CD30 Car-T cell therapy strategy and test its effect in PTCL. Method: The extracellular fragment gene sequences of CD30 were obtained from CD30 positive tumor tissues of PTCL patients and cloned into a recombinant plasmid to obtained CD30 protein. Then, the CD30 targeting single-chain antibody fragment (scFv) gene sequences were obtained from CD30 positive monoclonal hybridoma cell. The T cells were stimulated by using OKT3, CD28 and IL-2 from peripheral blood mononuclear cell (PBMC), then transfected with CD30 Lenti-CAR. The numbers of CD30 positive T cells were counted by flow cytometry after 14 days culture. The cytotoxic function of Car-T cells to CD30+ PTCL cells were detected using chromium release assay (CRA) and real-time cell electronic sensing assay (xCelligence station, ACEA biosciences). Results: The SDS-PAGE data shows that the molecular weight of CD30 protein was 45KDa, and the mass spectrometry data indicate that amino acid sequences were consistent with the prediction. The flow cytometry data shows 35% CD30 positive Car-T cells after transfection with CD30 Lenti-Car at day of 14. The Car-T cells can significantly inhibit the 299 cells proliferation at 20:1 effector-to-target ratios after 12 hours. The real-time cell electronic sensing data shows the Car-T cells can significantly inhibit the 299 cells proliferation at effector-to-target ratios of 5:1, 10:1, 20:1 and 40:1 after 6 hours co-culture. Conclusions: In summary, we have shown a strategy for treating CD30 positive PTCL with Car-T cell therapy. We obtained a CD30 antibody with high specificity and affinity though immunized of mouse by CD30 antigen which sequences comes from the CD30 positive PTCL tissue. We also established real-time cell electronic sensing method to assessing cytotoxic function of Car-T cells and Car-T cells show high efficiency to cytotoxic of Karpas 299 cells. Therefore, this new CD30 Car-T cells therapy strategy may provide an effective and safe treatment of PTCL. Note: This abstract was not presented at the meeting. Citation Format: Yang Wu, Dan Chen, Rong Ma, Jun-ying Zhang, Yuan Zhang, Hai-xia Cao, Zhuo Wang, Chang-wen Jing, Si-wen Liu, Ji-feng Feng, Jian-Zhong Wu. The new therapy strategy for treatment of peripheral T cell lymphomas: CD30-targeted CAR-modified T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1444.

The fatty acid synthase (FAS) gene is significantly up-regulated in various types of cancers, and blocking the FAS expression results in apoptosis of tumor cells. Therefore, FAS is considered to be an attractive target for anticancer therapy. However, the molecular mechanism by which the FAS gene is up-regulated in tumor cells is poorly understood. We found that FAS was significantly up-regulated by hypoxia, which was also accompanied by reactive oxygen species (ROS) generation in human breast cancer cell lines. The FAS expression was also activated by H2O2, whereas N-acetyl-l-cystein, a ROS inhibitor, suppressed the expression. We also found that the hypoxia significantly up-regulated sterol regulatory–element binding protein (SREBP)-1, the major transcriptional regulator of the FAS gene, via phosphorylation of Akt followed by activation of hypoxia-inducible factor 1 (HIF1). Moreover, our results of reporter assay and chromatin immunoprecipitation analysis indicate that SREBP-1 strongly bound to the SREBP binding site/E-box sequence on the FAS promoter under hypoxia. In our xenograft mouse model, FAS was strongly expressed in the hypoxic regions of the tumor. In addition, our results of immunohistochemical analysis for human breast tumor specimens indicate that the expressions of both FAS and SREBP-1 were colocalized with hypoxic regions in the tumors. Furthermore, we found that hypoxia-induced chemoresistance to cyclophosphamide was partially blocked by a combination of FAS inhibitor and cyclophosphamide. Taken together, our results indicate that FAS gene is up-regulated by hypoxia via activation of the Akt and HIF1 followed by the induction of the SREBP-1 gene, and that hypoxia-induced chemoresistance is partly due to the up-regulation of FAS. [Cancer Res 2008;68(4):1003–11]

The radiomimetic enediyne C-1027 induces almost exclusively DNA double-strand breaks (DSB) and is extremely cytotoxic. Unique among radiomimetics, ataxia-telangiectasia mutated (ATM) is dispensable for cellular responses to C-1027-induced DNA damage. This study explores the biological activity of three recently bioengineered C-1027 analogues: 7″-desmethyl-C-1027 (desmethyl), 20′-deschloro-C-1027 (deschloro), and 22′-deshydroxy-C-1027 (deshydroxy). Each compound maintains the characteristic ability of radiomimetics to cleave DNA in cell-free systems, varying in activity from 2-fold (deschloro) to 55-fold (desmethyl) less than C-1027. The induction of cellular DNA breaks based on pulsed field gel electrophoresis, comet analysis, and γH2AX activation was in the same rank order as cell-free DNA break induction, although the amount of breaks induced by desmethyl is greatly reduced compared with the other analogues. Despite the disparity in inducing DNA DSBs, all of the analogues produced G2-M cell cycle arrest and activated DNA DSB damage response proteins, such as p53-Ser15 and Chk2-Thr68, at concentrations in concordance with their ability to inhibit cell growth. Interestingly, of the three analogues, only the desmethyl-induced DNA damage response was similar to C-1027, as it did not cause hypersensitive cell growth inhibition in the absence of ATM nor require the kinase to phosphorylate p53 or Chk2. These findings show that simple modifications of the chromophore of C-1027 can result in varied induction of, and cellular response to, DNA DSBs. [Cancer Res 2007;67(2):773–81]

Cancer cells tend to utilize aerobic glycolysis even under normoxic conditions, commonly called the “Warburg effect.” Aerobic glycolysis often directly correlates with malignancy, but its purpose, if any, in metastasis remains unclear. When wild-type KISS1 metastasis suppressor is expressed, aerobic glycolysis decreases and oxidative phosphorylation predominates. However, when KISS1 is missing the secretion signal peptide (ΔSS), invasion and metastasis are no longer suppressed and cells continue to metabolize using aerobic glycolysis. KISS1-expressing cells have 30% to 50% more mitochondrial mass than ΔSS-expressing cells, which are accompanied by correspondingly increased mitochondrial gene expression and higher expression of PGC1α, a master coactivator that regulates mitochondrial mass and metabolism. PGC1α-mediated downstream pathways (i.e., fatty acid synthesis and β-oxidation) are differentially regulated by KISS1, apparently reliant upon direct KISS1 interaction with NRF1, a major transcription factor involved in mitochondrial biogenesis. Since the downstream effects could be reversed using short hairpin RNA to KISS1 or PGC1α, these data appear to directly connect changes in mitochondria mass, cellular glucose metabolism, and metastasis. Cancer Res; 74(3); 954–63. ©2013 AACR.

The molecular mechanisms that operate within the organ microenvironment to support metastatic progression remain unclear. Here, we report that upregulation of hyaluronan synthase 2 (HAS2) occurs in highly metastatic breast cancer stem–like cells (CSC) defined by CD44+/CD24−/ESA+ phenotype, where it plays a critical role in the generation of a prometastatic microenvironment in breast cancer. HAS2 was critical for the interaction of CSCs with tumor-associated macrophages (TAM), leading to enhanced secretion of platelet-derived growth factor-BB from TAMs, which then activated stromal cells and enhanced CSC self-renewal. Loss of HAS2 in CSCs or treatment with 4-methylumbelliferone, an inhibitor of HAS, which blocks hyaluronan production, drastically reduced the incidence and growth of metastatic lesions in vitro or in vivo, respectively. Taken together, our findings show a critical role of HAS2 in the development of a prometastatic microenvironment and suggest that HAS2 inhibitors can act as antimetastatic agents that disrupt a paracrine growth factor loop within this microenvironment. Cancer Res; 72(2); 537–47. ©2011 AACR.

RhoC is a member of the Ras-homologous family of genes which have been implicated in tumorigenesis and tumor progression. However, the exact role of RhoC is controversial and is yet to be clarified. We have examined the effect of RhoC on prostate tumor cells and found that RhoC had no effect on cell proliferation in vitro or on tumor growth in mice. However, RhoC significantly enhanced the metastatic ability of the tumor cells in these animals, suggesting that RhoC affects only the metastasis but not the growth of prostate tumor cells. The results of our immunohistochemical analyses on tumor specimens from 63 patients with prostate cancer indicate that RhoC expression had no significant correlation with Gleason grade. However, the expression of RhoC showed significant positive correlation with both lymph node and distant metastasis, and it was inversely correlated with patient survival. We also found that RhoC significantly augmented the invasion and motility of prostate tumor cells by activating matrix metalloproteinases 2 and 9 (MMP2 and MMP9) in vitro. The results of our antibody array analysis for signal molecules revealed that RhoC significantly activated kinases including mitogen-activated protein kinase (MAPK), focal adhesion kinase (FAK), Akt, and Pyk2. Inhibition of Pyk2 kinase blocked the RhoC-dependent activation of FAK, MAPK, and Akt, followed by the suppression of MMP2 and MMP9. Inhibitors of both MAPK and Akt also significantly blocked the activities of these MMPs. Therefore, our results indicate that RhoC promotes tumor metastasis in prostate cancer by sequential activation of Pyk2, FAK, MAPK, and Akt followed by the up-regulation of MMP2 and MMP9, which results in the stimulation of invasiveness of tumor cells. [Cancer Res 2008;68(18):7613–20]

The tumor metastasis suppressor gene Drg-1 has been shown to suppress metastasis without affecting tumorigenicity in immunodeficient mouse models of prostate and colon cancer. Expression of Drg-1 has also been found to have a significant inverse correlation with metastasis or invasiveness in various types of human cancer. However, how Drg-1 exerts its metastasis suppressor function remains unknown. In the present study, to elucidate the mechanism of action of the Drg-1 gene, we did a microarray analysis and found that induction of Drg-1 significantly inhibited the expression of activating transcription factor (ATF) 3, a member of the ATF/cyclic AMP–responsive element binding protein family of transcription factors. We also showed that Drg-1 attenuated the endogenous level of ATF3 mRNA and protein in prostate cancer cells, whereas Drg-1 small interfering RNA up-regulated the ATF3 expression. Furthermore, Drg-1 suppressed the promoter activity of the ATF3 gene, indicating that Drg-1 regulates ATF3 expression at the transcriptional level. Our immunohistochemical analysis on prostate cancer specimens revealed that nuclear expression of ATF3 was inversely correlated to Drg-1 expression and positively correlated to metastases. Consistently, we have found that ATF3 overexpression promoted invasiveness of prostate tumor cells in vitro, whereas Drg-1 suppressed the invasive ability of these cells. More importantly, overexpression of ATF3 in prostate cancer cells significantly enhanced spontaneous lung metastasis of these cells without affecting primary tumorigenicity in a severe combined immunodeficient mouse model. Taken together, our results strongly suggest that Drg-1 suppresses metastasis of prostate tumor cells, at least in part, by inhibiting the invasive ability of the cells via down-regulation of the expression of the ATF3 gene. (Cancer Res 2006; 66(24): 11983-90)

Considering the enormous energy requirements and stresses of the metastatic cascade, an interrelationship with aerobic glycolysis seems intuitive, but has not been definitively established. The purpose of these studies was to determine whether there is a relationship between KISS1 metastasis suppression and metabolism. Wild-type KISS1 expressed in human melanoma cells were metastasis suppressed and took up less glucose and produced less lactate, corresponding to higher pH[Ex]. Metabolism and metastasis changes did not occur when KISS1 was missing the secretion signal peptide (ΔSS). Changes in glucose transport and key glycolytic enzymes did not consistently correlate with metabolic changes; however, V-ATPase, which promotes extracellular acidification, invasion and metastasis, appears to be involved in KISS1-mediated metabolic changes. Also corresponding with the shift from glycolysis to oxidative phosphorylation, KISS1-expressing cells have 30-50% more mitochondrial mass accompanied increased higher expression of PPARγ co-activator 1α (PGC1α), a master regulator of mitochondrial biogenesis. PGC1α-mediated downstream pathways (i.e. fatty acid synthesis and β-oxidation) are differentially regulated by KISS1, apparently reliant upon direct KISS1 interaction with Nuclear Respiratory Factor 1 (NRF1), a major transcription factor involved in mitochondrial biogenesis. KISS1 does not affect PGC1α mRNA expression but stabilizes the protein through interaction with the ubiquitin-like protein, UBQLN1. To test whether a KISS1- PGC1α axis is critical for metastasis suppressor function, shRNA to KISS1 or PGC1α was introduced into KISS1-expressing cells. Metabolic changes and suppression of invasion and migration were reversed. Importantly, knock-down of PGC1α abolished KISS1-mediated metastasis suppression in vivo, strongly suggesting that PGC1α is an essential downstream mediator of KISS1 as a metastasis suppressor. Taken together, these data define a novel signaling pathway controlling metabolism and metastasis. Moreover, these data appear to directly connect changes in aerobic glycolysis, mitochondrial metabolism, and cancer metastasis. Citation Format: Wen Liu, Benjamin Beck, Kedar S. Vaidya, Kevin T. Nash, Scott W. Ballinger, Danny R. Welch. The KISS1 metastasis suppressor appears to integrate glycolysis, mitochondrial biogenesis and metastasis via regulation of a PGC1α 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 126. doi:10.1158/1538-7445.AM2014-126

Head and neck squamous cell carcinoma (HNSCC) is one of the serious life-threatening diseases in the world. In addition to surgery, the conventional treatments for oral cancer includes radiation and chemotherapy, which usually act by induction of DNA damage. However, approximately one-third of treated patients will experience local or regional recurrence. G9a is a histone methyltransferase responsible for mono- or di-methylation of lysine 9 in histone H3. Our recent work has demonstrated that G9a level is highly correlated with aggressiveness and poor prognosis of lung, ovarian and colon cancer patients in Taiwanese population. Here, we found that G9a expression in HNSCC is upregulated and positively correlated with poor prognosis. Moreover, knockdown of G9a resulted in decrease cell survival and cologenic capacity following treating with chemotherapeutic drug, hydroxyurea and etoposide. Futhermore, depletion of G9a caused sustained phosphorylation of γ-H2AX after etoposide treatment. Together, We suggest that G9a may contribute to HNSCC proliferation by improvement of DNA double break repair. Our study paves the way for exploring the blockade of G9a overexpression as a novel approach for the prevention and treatment of HNSCC. Citation Format: Chia-Wen Liu. The role of histone methyltransferase G9a in chemotherapeutic drugs-induced DNA damage. [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 419. doi:10.1158/1538-7445.AM2014-419

Tissue inhibitors of metalloproteinases (TIMPs) inhibit matrix metalloproteinases and some members of a disintegrin and metalloproteinase domain (ADAM) family. In addition, recent studies unveiled novel functions of TIMPs in the regulation of apoptosis. TIMP-1 inhibits intrinsic apoptosis by inducing TIMP-1 specific cell survival pathways involving focal adhesion kinase (FAK). TIMP-3, however, was shown to enhance extrinsic cell death by inhibiting the shedding of the cell surface death receptors mediated by tumor necrosis factor-α converting enzymes (TACE/ADAM-17). Here, we examined whether TIMP-1, an inhibitor of some of the ADAM family members, enhances the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)–induced extrinsic apoptotic pathway. Surprisingly, we found that TIMP-1 effectively protects human breast epithelial cells from TRAIL-induced apoptosis, demonstrating opposite roles of TIMP-1 and TIMP-3 for the regulation of extrinsic apoptosis. TIMP-1 inhibition of TRAIL-induced apoptosis does not depend on its ability to inhibit matrix metalloproteinases or ADAM activities and is unrelated to its ability to stabilize active or decoy death receptors. Importantly, inhibition of PI 3-kinase signaling by wortmannin and down-regulation of FAK expression using siRNA significantly diminish TIMP-1 protection of human breast epithelial cells against TRAIL-induced extrinsic apoptosis. In addition, the in vitro three-dimensional culture studies showed that TIMP-1 inhibits lumen formation and apoptosis during morphogenesis of MCF10A acini. Taken together, these studies suggest that TIMP-1 may exert oncogenic activity in breast cancer through inhibition of both intrinsic and extrinsic apoptosis involving the FAK survival signal transduction pathway.

Oral cancer is one of the serious life-threatening diseases in the world. In addition to surgery, the conventional treatment for oral cancer includes radiation and anti-cancer drugs, which usually act by induction of DNA damage. However, approximately one-third of treated patients will experience local or regional recurrence. G9a is a histone methyltransferase responsible for mono- or dimethylation of lysine 9 in histone H3. Our recent work has demonstrated that G9a level is highly correlated with aggressiveness and poor prognosis of lung, ovarian and colon cancer patients in Taiwanese population. Here, we report that knockdown of G9a resulted in decrease cell survival and cologenic capacity following DNA damage induced independently by etoposide and ionizing radiation. Depletion of G9a caused sustained phosphorylation of γ-H2AX after etoposide treatment. Together, our data suggest that a novel function of G9a may increase the DNA repair efficiency and promote the oral cancer cells recovery and survival from the DNA damage agents. We suggest that G9a may contribute to oral cancer proliferation by improvement of homologous recombination repair of DNA double break. Our study paves the way for exploring the blockade of G9a overexpression as a novel approach for the prevention and treatment of oral cancer. Citation Format: Chia-Wen Liu, Shih-Ting Cha, Ching-Ting Tan, Min-Liang Kuo. Histone methyltransferase G9a promotes the oral cancer cells recovery from drug-induced DNA damage. [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 4063. doi:10.1158/1538-7445.AM2013-4063

Cancer cells tend to utilize aerobic glycolysis even under normoxic conditions, which is commonly called the “Warburg Effect.” Aerobic glycolysis often directly correlates with malignant potential. Though its purpose remains unclear, the “Warburg Effect” is thought to confer advantages to proliferation, survival and dissemination to cancer cells by increasing uptake of nutrients into biomass. KISS1 protein is secreted and proteolytically cleaved into kisspeptins (KP) that block the colonization of disseminated metastatic C8161.9 human melanoma cells at secondary sites. In this study, we hypothesized that KISS1 metastasis suppression occurs via regulation of aerobic glycolysis. Comparison of bioenergetic and metabolic aspects of glucose metabolism showed that all KISS1-secreting clones were less invasive, took up less glucose, produced less lactate which corresponds to higher pH[Ex], effects which were reversed when cells were transduced with shRNA to KISS1. The metabolism, invasion, and metastasis changes did not occur when KISS1 was missing the signal peptide (ΔSS). Utilizing a Seahorse bioanalyzer, KISS1, but not ΔSS cells showed significantly decreased extracellular acidification rates, increased O2 consumption and elevated mitochondria reserve capacity, an indicator of mitochondrial condition and a parameter thought to improve the cells’ ability to cope with oxidative stress. KISS1-expressing cells have 30-50% more mitochondria compared to vector or ΔSS-expressing cells. Increased mitochondrial mass was accompanied by significantly increased expression of mitochondrial genes involved in apoptosis and mitophagy, protein processing and trafficking. Increased mitochondrial mass correlated with higher PGC1α considered to be a master co-activator that regulates mitochondrial mass and metabolism. Interestingly, KISS1 differentially affects PGC1α-mediated downstream pathways, i.e. fatty acid synthesis and β-oxidation. KISS1-mediated up-regulation of mitochondria biogenesis appears to rely on KISS1 interaction with NRF1, a major transcription factor of mitochondria biogenesis. KP10 (which can activate the KISS1 receptor) does not alter pH[Ex] since the metastatic tumor cells do not express KISS1R. This paradox - metastasis and metabolic changes require secretion, but responding cells do not have the receptor - raises questions regarding the mechanism. Nonetheless, these data appear to directly connect changes in mitochondria mass, cellular glucose metabolism and metastasis. [Support: CA134581, Natl. Fndn. Cancer Res., Komen SAC110037]. Citation Format: Wen Liu, Benjamin H. Beck, Kedar S. Vaidya, Kevin T. Nash, Anne R. Diers, Kyle P. Feeley, Aimee Landar, Scott W. Ballinger, Danny R. Welch. The KISS1 metastasis suppressor appears to reverse the Warburg effect by enhancing mitochondria biogenesis. [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 3866. doi:10.1158/1538-7445.AM2013-3866

The molecular mechanism of metastatic progression, particularly the roles of microenvironment of the metastasized organs, is as yet poorly understood. In this study, we have isolated cancer stem cell-like cells (CSC) from human breast cancer cell lines, MDA-MB231, MDA231BoM and MDA231BrM. CSC were isolated from each line using surface markers (CD24-, CD44+, ESA+) and their tumor initiating abilities were tested in animal. We then performed an analysis of global expression profile for these CSCs using the Affymetrix expression array and found that among all up-regulated genes, two genes (HAS2 and MMP1) were significantly correlated with overall- and metastasis-free survival when they were cross-examined in GEO database. To further examine the role of HAS2 in tumor metastasis in vivo, we prepared CSC from MDA231BoM with or without carrying a shRNA expression vector for HAS2. The CSC were then implanted into nude mice by intra-cardiac injection followed by oral administration of 4MU, a specific inhibitor of HAS genes. We found that the injection of CSC of MDA231BoM which expresses shRNA to HAS2 significantly improved the survival rate of animals. In addition, administration of 4MU significantly suppressed tumor metastasis of CSC, suggesting that inhibition of HAS2 can indeed reduce the incidence of metastasis in vivo. We also examined the effect of tumor associated macrophage (TAM) on growth of CSC in animals by injecting CSC of MDA231-BoM with or without TAM into the tibial bone. We found that co-injection of TAM with CSC significantly augmented the growth of tumor in their legs. Moreover, the interaction between cancer cell and TAMs enhanced secretion of PDGF-BB from TAMs which then activated stromal cells and in turn enhanced self-renewal of CSCs by secretion of FGF9 and FGF7. We also found that treatment of bone marrow stem cell (BMSC) with PDGF followed by co-culture of CSC significantly promoted proliferation of CSC. Collectively, our results suggest that TAM and BMSC promote self-renewal of CSC in bone microenvironment via direct interaction between hyaluronan and CD44. Our results also provide strong rationale to target HAS2 by using 4MU in order to block the metastatic progression of CSC in breast cancer. Citation Format: Sambad Sharma, Hiroshi Okuda, Fei Xing, Aya Kobayashi, Misako Watabe, Puspa R. Pandey, Sudha K. Pai, Wen Liu, Koji Fukuda, Megumi Iiizumi, Kounosuke Watabe. HAS2 promotes tumor progression by stimulating interaction of cancer stem-like cells with tumor associated macrophage and stromal cells through a paracrine loop of growth factors in the bone. [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 3865. doi:10.1158/1538-7445.AM2013-3865

The molecular mechanism of metastatic progression, particularly the roles of microenvironment of the metastasized organs, is as yet poorly understood. In this study, we have isolated cancer stem cell-like cells (CSC) from human breast cancer cell lines, MDA-MB231, MDA231BoM and MDA231BrM. CSC were isolated from each line using surface markers (CD24-, CD44+, ESA+) and their tumor initiating abilities were tested in animal. We then performed an analysis of global expression profile for these CSCs using the Affymetrix expression array and found that among all up-regulated genes, two genes (HAS2 and MMP1) were significantly correlated with overall- and metastasis-free survival when they were cross-examined in GEO database. To further examine the role of HAS2 in tumor metastasis in vivo, we prepared CSC from MDA231BoM with or without carrying a shRNA expression vector for HAS2. The CSC were then implanted into nude mice by intra-cardiac injection followed by oral administration of 4MU, a specific inhibitor of HAS genes. We found that the injection of CSC of MDA231BoM which expresses shRNA to HAS2 significantly improved the survival rate of animals. In addition, administration of 4MU significantly suppressed tumor metastasis of CSC, suggesting that inhibition of HAS2 can indeed reduce the incidence of metastasis in vivo. We also examined the effect of tumor associated macrophage (TAM) on growth of CSC in animals by injecting CSC of MDA231-BoM with or without TAM into the tibial bone. We found that co-injection of TAM with CSC significantly augmented the growth of tumor in their legs. Moreover, the interaction between cancer cell and TAMs enhanced secretion of PDGF-BB from TAMs which then activated stromal cells and in turn enhanced self-renewal of CSCs by secretion of FGF9 and FGF7. We also found that treatment of bone marrow stem cell (BMSC) with PDGF followed by co-culture of CSC significantly promoted proliferation of CSC. Collectively, our results suggest that TAM and BMSC promote self-renewal of CSC in bone microenvironment via direct interaction between hyaluronan and CD44. Our results also provide strong rationale to target HAS2 by using 4MU in order to block the metastatic progression of CSC in breast 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 3323. doi:1538-7445.AM2012-3323

It is of paramount interest to delineate the underlining molecular pathways of cancer metastasis in order to identify a novel therapeutic target for this devastating disease. Previously, we identified NDRG1 as a tumor metastasis suppressor gene and found that it is negatively involved in metastatic progression of prostate and breast cancer. Herein, we sought to determine how NDRG1 gene exerts its metastasis suppressor function. We used the yeast two-hybrid system to identify proteins interacting with NDRG1, and also the phospho-site profiling system to search for potential signaling pathway involved in NDRG1 function. We found that NDRG1 interacted with Wnt receptor, LRP6, and blocked its phosphorylation. Moreover, NDRG1 induced phosphorylated- activation of GSK3β (Tyr279/216), an essential negative regulator of Wnt pathway, followed by inhibition of β-catenin/TCF transcriptional activity. Further investigations revealed that NDRG1 inhibited the expression of ATF3, a potent metastasis promoter, through Wnt- β-catenin/TCF signaling. Consistently, the results of our immunohisochemical studies revealed an inverse correlation between NDRG1 expression and ATF3 and membrane β-catenin, respectively, while ATF3 expression was significantly correlated with cytoplasm/nuclear expression of β-catenin in prostate cancer patients. When we assessed the possible steps of metastasis cascade that were affected by NDRG1, we found that NDRG1 dramatically inhibited Wnt-induced EMT of tumor cells. Furthermore, NDRG1 not only significantly reduced local invasive ability of tumor cells, but also impaired the later steps of invasion-metastasis cascade including infiltration, initial survival and extravasation in the mice lung parenchyma. To determine whether NDRG1 indeed suppresses metastasis via the inhibition of Wnt pathway in vivo, we isolated tumor cells from a MMTV-Wnt1 mouse and established a series of cell lines with or without NDRG1 expression in combination with a dominant active Wnt signaling. Our results indicate that NDRG1 significantly decreased metastasis while it did not affect the primary tumor growth. However, a dominant active form of β-catenin effectively overcame the metastasis-suppressive effect of NDRG1. Importantly, restoring NDRG1 expression by a small molecule compound significantly suppressed the capability of otherwise highly metastatic tumor cells to thrive in circulation and distant organs in animal models. Finally, using a combined expression status of Wnt and NDRG1 as a signature, we could stratify patients who have significant higher relapse or metastasis incidence than others in clinical databases. Taken together, our results revealed a novel mechanism of metastasis suppression by which NDRG1 suppresses pleiotropic effects of Wnt signaling on metastatic cascade through impeding the upstream activation of Wnt co-receptor LRP6. 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 3419. doi:1538-7445.AM2012-3419

It has been suggested that NF-κB triggers both the onset and resolution of inflammation by governing the expression of genes essential in activating malignancy-promoting signaling pathways. Members of the NF-κB activation pathway include tumor necrosis factor (TNF), several interleukins (IL), TNF receptor associated factors (TRAFs), and toll-like receptors (TLR). These molecules are involved in cell proliferation, differentiation, apoptosis and response to proinflammatory cytokines and mediating the production of cytokines necessary for the development of effective immunity. Common single nucleotide polymorphisms (SNP) in TNF, IL, TRAF, TLR and the main units of NF-κB genes were assessed for the association with prostate cancer (PrCA) risk in a clinic-based study of Caucasian and African-American cases and controls. A total of 709 tagSNPs (with minor allele frequency > = 5% and r2>=0.8) in 54 genes were genotyped in 903 Caucasians (552 cases and 351 controls) and 174 African-Americans (74 cases and 100 controls) using a custom Illumina Infinium II ® assay. Logistic regression models were used to examine each tagSNP with PrCA (adjusting for age and family history of prostate cancer and using an additive model for the SNP). Empirical p-values were generated by permutation (using 10,000 replicates) to correct for multiple comparisons. Statistically significant associations were observed in Caucasians at the NFkBIL1 and IL1R1 genes. The strongest evidence for association came at a polymorphism in the promoter region of the NFkBIL1 gene (rs3219184) (OR = 0.51 [95% CI 0.35-0.74], p = 0.0005), and a polymorphism in the 3’ flanking region of the IL1R1 gene (rs3917332) (OR = 1.60 [95% CI 1.23-2.09], p = 0.0005). We conclude that common variations in NFkBIL1 and IL1R1 might play important roles in PrCA susceptibility. 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 2771. doi:10.1158/1538-7445.AM2011-2771

Notch signaling is often and aberrantly activated by hypoxia during tumor progression; however, the exact pathological role of hypoxia-induced Notch signaling in tumor metastasis is as yet poorly understood. In this study, we aimed to define the mechanism of Notch ligand activation by hypoxia in both primary tumor and bone stromal cells in the metastatic niche and to clarify their roles in tumor progression. We have analyzed the expression profiles of various Notch liagnds in 779 breast cancer patients in GEO database and found that the expression of Jagged2 among all five ligands is most significantly correlated with the overall- and metastasis-free survival of breast cancer patients. The results of our immunohistochemical (IHC) analysis for Jagged2 in 61 clinical samples also revealed that both Jagged2 and Notch signaling were strongly up-regulated at the hypoxic invasive front. Activation of Jagged2 by hypoxia in tumor cells induced EMT and also promoted cell survival in vitro. Notably, a γ-secretase inhibitor significantly blocked Notch-mediated invasion and survival under hypoxia by promoting expression of E-cadherin and inhibiting AKT phosphorylation. Importantly, Jagged2 was also found to be up-regulated in bone marrow stroma under hypoxia and promoted the growth of cancer stem-like cells by activating their Notch signaling. Therefore, hypoxia-induced Jagged2 activation in both tumor invasive front and normal bone stroma plays a critical role in tumor progression and metastasis, and Jagged2 is considered to be a valuable prognostic marker and may serve as a novel therapeutic target for metastatic breast cancer. 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 1028. doi:10.1158/1538-7445.AM2011-1028

Multiple dys-regulated cell signaling pathways and extensive cross-talks, which can be depicted via protein-protein interactions (PPIs), among individual signaling pathways, characterize cancer development and progression. However, systematic analysis of the cross-talk events from distinct pathways via PPIs remains fragmentary, and the reliability of PPI datasets, particularly at the cellular level, remains to be vigorously examined. Here, we first build the cross-talk pathway map of heptocellular carcinoma (HCC) through overexpression patterns in HCC among 23 pathways. By detecting endogenous PPIs in two HCC cell lines, Huh7 (low invasive cells) and Mahlavu (high invasive cells), we validate 50 PPI events within pathway and 19 PPI-mediated cross-talk events among 23 pathways in HCC. Not surprisingly, many distinct PPIs are present between Huh7 and Mahlavu, providing the opportunity to characterize tumor invasion. By analyzing topology of cross-talk PPI network, CRKL has a pivotal role in cross-talk pathway. This initial attempt to map the cross-talk pathways in HCC may represent the first step in deciphering hepatocarcinogenesis at the systems level and might reveal a novel strategy for HCC treatment. 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 4929. doi:10.1158/1538-7445.AM2011-4929

Elevated level of lipogenic enzymes and overall lipogenesis have been reported in a wide variety of cancers, and blocking the lipogenic pathway by chemical inhibitors or RNA interference causes tumor cell death by apoptosis. In this study, we investigated the role of lipogenesis in cancer stem-like cells (CSCs) in ductal carcinoma in situ (DCIS) which is considered as an early stage of breast tumorigenesis. CSCs were isolated using cell surface markers (CD24−/CD44+/ESA+) from the MCF10DCIS. Com (DCIS.com) cell line and transplanted into the mammary fat pad of nude mice. The results of our limiting dilution analysis indicate that CSCs had a significantly higher ability of initiating DCIS in the animals. We then examined the expression profile of various lipogenic genes using an expression microarray and found that CSCs from DCIS.com showed significantly higher level of ACLY, ACC-1 and FAS than the normal non-tumorigenic stem-like cells. The result was also confirmed by qRT-PCR and Western blot. We also examined the expression of these genes in clinical specimens of DCIS by immunohistochemistry and found that these genes are indeed significantly upregulated. Furthermore, we found SREBP1, the master regulator of lipogenic genes, was also significantly upregulated in DCIS clinical specimens as well as in DCIS.com cells and inhibition of SREBP1 significantly downregulated the expression of ACLY, ACC-1 and FAS. These results suggest that the lipogenic enzymes play critical roles in initiation and maintenance of DCIS. We then examined the effects of Resveratrol, a natural polyphenol, on CSCs isolated from DCIS.com. We found that Resveratrol reduced the total lipid content of DCIS CSCs by inhibiting lipogenic genes and induced a series of pro-apoptotic genes causing the death of DCIS CSCs. Resveratrol also hindered the stemness of the DCIS CSCs by inhibiting its mammosphere forming ability. When DCIS CSCs was transplanted into mammary fat pad of nude mice followed by treatment with Resveratrol, we observed that Resveratrol indeed significantly suppressed the formation of DCIS by downregulating lipogenic genes and by upregulating pro-apoptotic genes, DAPK2 and BNIP3. Collectively, our results indicate that lipogenic genes ACLY, ACC-1, FAS and SREBP1 are significantly up regulated in DCIS CSCs at an early stage of breast tumorigenesis and they confer proliferative and anti-apoptotic capacity to CSCs. Anti-growth effect of Resveratrol on DCIS CSCs also provides us with a strong rationale to use this agent for chemo-prevention against DCIS. 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 979. doi:10.1158/1538-7445.AM2011-979

According to the recent cancer stem theory, metastatic cells must have stem cell-like capability for their self-renewal and invasive ability. Understanding how the cancer stem cells adapt themselves to the microenvironment of the distant organs and how the “niche” at the metastatic site promotes the proliferation of these cells is of paramount importance to develop more effective therapeutic strategy for metastatic disease. In this study, we have isolated cancer stem cell-like cells (CSC) from human breast cancer cell lines, MDA-MB231, MDA231BoM and MDA231BrM. The latter two cell lines are derivatives of MDA-MB231 and they preferentially metastasize to bone (MDA231BoM) or brain (MDA231BrM) when they are transplanted into mice. CSC were isolated from each line using surface markers (CD24-, CD44+, ESA+) and their tumor initiating abilities were tested in animal. We then performed an analysis of global expression profile for these CSCs using the Affymetrix expression array and found that among all up-regulated genes, two genes (HAS2 and MMP1) were significantly correlated with overall- and metastasis-free survival when they were cross-examined in GEO database. To further examine the role of HAS2 in tumor metastasis in vivo, we prepared CSC from MDA231BoM which carries shRNA expression vector for HAS2. The CSC were then implanted into nude mice by intra-cardiac injection followed by oral administration of 4MU, a specific inhibitor of HAS genes. We found that the injection of CSC of MDA231BoM which expresses shRNA significantly improved the survival rate of animals. In addition, administration of 4MU significantly suppressed tumor metastasis of CSC, suggesting that inhibition of HAS2 can indeed reduce the incidence of metastasis in vivo. We also examined the effect of tumor associated macrophage (TAM) on growth of CSC in animals by injecting CSC of MDA231-BoM with or without TAM into the tibial bone. We found that co-injection of TAM with CSC significantly augmented the growth of tumor in their legs. These results strongly support the idea that TAM plays a critical role in CSC growth in breast cancer. To understand how TAM promotes CSC proliferation, we attempted to identify secretory factor(s) from TAM by using a cytokine/growth factor antibody array system. We found that the secretion of PDGF from TAM was significantly augmented in conditioned medium of co-culture of CSC and TAM. Furthermore, we found that treatment of bone marrow stem cell (BMSC) with PDGF followed by co-culture of CSC significantly promoted proliferation of CSC. Collectively, our results suggest that TAM and BMSC promote self-renewal of CSC in bone microenvironment via direct interaction between hyaluronan and CD44. Our results also provide strong rationale to target HAS2 by using 4MU in order to block the metastatic progression of CSC in breast cancer. 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 3409. doi:10.1158/1538-7445.AM2011-3409

Signal transduction pathways at cellular level require massive protein-protein interactions (PPIs) in response to different environmental cues. Diverse experimental techniques for detecting PPIs have been developed; however, the large amount of data accumulated from various sources has posed a grand challenge in data reliability for PPI. To evaluate the role of PPIs within signaling pathways, we have systematic profiled PPI residing in 28 KEGG pathways, for example mTOR, MAPK, Wnt, VEGF, and Hedgehog, and 3 cancer-related pathways stored in KEGG (http://www.genome.jp/kegg/). Of ∼60000 human PPI collected, 2786 PPI can be detected within targeted 14 pathways. To validate these PPIs, we used a newly developed Proximity Ligation Assay, which detects endogenous PPI through a pair of antibodies that bind to proteins in close proximity. Each PPI can be made visible through DNA amplification and quantified by counting fluorescent dots in the cell. Of 1,204 PPIs examined, 557 PPIs can be confirmed in HeLa cells. Specifically, 383 out of 557 validated PPIs residing within pathways in cancer and mTOR, Wnt and MAPK pathways are considered novel “links” based on annotations of KEGG. Moreover, 79 validated PPIs are cross-talk events among 35 signaling transduction and cancer related pathways. Surprisingly, only 16 PPIs (out of 557 confirmed PPIs) are highly correlated in 7 sets of cancer cells from NCI60 microarray dataset. In this large scale PPI profiling, 41 apoptosis related PPIs were further tested in 3 cancer cell lines to explore the possibility for drug screening. These systematic analyses of endogenous PPI pairs within and between pathways may set the foundation to disrupt the signaling pathways by targeting PPIs in cancer drug discovery. 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 1976. doi:10.1158/1538-7445.AM2011-1976

The presence of CTCs in the peripheral blood, as detected by the FDA-approved CellSearch® CTC Kit, is associated with decreased overall and progression free survival in patients with metastatic breast, prostate and colorectal cancers. In addition to CTC enumeration, CellSearch® CTC Kit provides a unique tool for CTC biomarker analysis in cancers of epithelial origin (CD45—, EpCAM+, and cytokeratins (CK) 8,18-positive and/or 19-positive). Considerable cell loss is suspected to occur at many of the automated steps of the CellSearch® CTC process, resulting in undermined recovery of CTCs. Herein, we demonstrate an improved, highly sensitive approach for CTC enumeration, which is based on the CellSearch® CTC Profile Kit and immunofluorescent analysis using LSC. The initial blood processing was conducted using the CellSearch® CTC Profile Kit from Veridex and the CellTracks® AutoPrep® system, which allowed recovery of the EpCAM+ cells directly from the instrument. Next, EpCAM+ cell population was subjected to manual immunofluorescent labeling with antibodies against CK and CD45, and CTCs were enumerated by LSC, based on the standard CellSearch® CTC criteria. When we compared this new method to standard CTC enumeration in patients with various cancer types including head and neck, renal cell, basal cell, prostate, non-small cell lung, sebaceous gland and ovarian cancers, we consistently detected higher CTC counts in samples analyzed with the new method. Indeed, 88% of patients (79 out of 90) were CTC-negative based on the standard analysis, while only 37% of patients were CTC-negative based on the CTC Profile Kit/LSC data. We next conducted a side-by-side comparison of the two techniques in patients with hepatocellular carcinoma (HCC, EpCAM-low) and prostate cancer (EpCAM+). CTC Profile Kit/LSC method demonstrated up to a 470% increased CTC recovery in the prostate cancer samples. Remarkably, it also allowed isolation of CTCs from the CTC-negative (as detected by the standard kit) HCC and prostate cancer samples. Finally, the new CTC enumeration method consistently yielded higher CTC counts in patients enrolled in a Phase I clinical trial of Bevacizumab and Cediranib. Importantly, our preliminary analysis indicated that changes in CTC counts from baseline to 24 h post-therapy, as measured by the new method, may correlate with changes in tumor size. In conclusion, we developed a new CellSearch® CTC Profile Kit/LSC-based method that offers higher sensitivity of CTC recovery and thus provides a broader capability for downstream molecular characterization. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2687.

Brain metastasis is found in around 20% of advanced breast cancer patients whose survival rates are generally less than one year after the diagnosis. Despite this clinical importance, the molecular basis of breast tumor metastasis to the brain is still virtually unknown. The goal of this project is to elucidate the role of brain microenvironment, particularly astrocytes, in breast tumor cell growth and invasion. Our analysis of existing microarray database indicate that interleukin-1β (IL-1β) is highly expressed in MDA-MB231BrM2a (231BrM) cell line which is capable of specifically metastasizing to the brain. IL-1β is known to have an ability of “activating” astrocytes that are often found in the metastatic niche, and therefore, it is speculated that the reactive astrocytes can promote growth and invasion of metastasized cells in the brain. To explore this possibility, we examined the effect of IL-1β and the conditioned medium of 231BrM cells on rat primary astrocytes in vitro. Our results of qRT-PCR and Western blot analysis indicate that IL-1β and the conditioned medium significantly up-regulated the Notch ligand, JAG1, in astrocytes, and that this up-regulation was abrogated by the NF-kB inhibitor, PDTC. To further examine the interaction of astrocytes and tumor cells in the brain, we intracardially injected 231BrM cells in nude mice, and tumor growth was monitored by Xenogen bioimager. After 4 weeks, metastatic tumor growth was observed at multiple sites of the brain, and the results of immunohistochemical analysis indicate that astrocytes adjacent to the metastatic lesions strongly expressed JAG1 and the reactive astrocyte marker, GFAP (Glial fibrillary acidic protein). These results strongly suggest that IL-1β secreted from 231BrM activates astrocytes and induces the expression of the Notch ligand, which in turn promotes the growth and invasion of metastasized breast tumor cells. To further test this hypothesis, we performed in vitro invasion assay by culturing astrocytes with or without the treatment of IL-1β on top of matrigel, followed by overlaying MDA-MB231 cells. We found that the IL-1β treatment of astrocytes significantly augments the invasive ability of MDA-MB231. We also introduced DNMAML (dominant negative mastermind) gene by lentivirus infection into 231BrM and found that the blocking of Notch signal by DNMAML failed to inhibit tumor growth but significantly down-regulated the expression of the MMPs (Matrix metallopeptidase) genes and decreased the invasive ability of 231BrM. These data strongly support our notion that a metastasized breast tumor cell in the brain secretes IL-1β and activates astrocytes which in turn up-regulates JAG1 and that the interaction of the activated astrocytes and tumor cells turns on Notch signaling in the tumor cells followed by promotion of tumor invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1413.

Prostate cancer is becoming a curable disease when it is diagnosed at an early stage. However, even those patients who have localized cancer and have been “successfully” treated with surgery often experience recurrent disease after many years of dormancy. In the case of prostate cancer, bone is the most common metastatic site which affects approximately 70% of patients with advanced disease. The growth of the tumor cells in the bone is generally slow and they often become dormant until an appropriate microenviroment is established for their re-growth. How these metastatic tumor cells become dormant and how they recur at the target organs is virtually unknown. To address this critical question, we have studied an interaction of prostate cancer cell (PC3mm) and bone stromal cell (HS5). Our data indicate that the conditioned medium of HS5 cells was able to induce growth arrest and senescence to prostate cancer cells through activation of p38 MAP kinase. Western blot, qRT-PCR and reporter assay showed that this effect was mediated by induction of the tumor metastasis suppressor gene, N-myc downstream regulated gene 1 (NDRG1). We found that this secretory factor is bone morphogenetic protein 7 (BMP7) and that a specific inhibitor of p38 significantly abrogated up-regulation of NDRG1 by BMP7, suggesting that BMP7 induces NDRG1 through activation of p38. We also found that BMP7 significantly activated p21 and that this effect was inhibited by siRNA to NDRG1. Importantly, our results indicate that BMP7 was able to induce senescence to PC3mm cell; however, this senescence was reversible and the tumor cells re-gained the ability of their growth upon withdrawal of BMP7, suggesting that BMP7-induced senescence is reversible. Notably, our results of the existing database analysis indicate that the expression level of BMPR2, one of the BMP7 receptors, showed inverse correlation with bone metastasis and recurrence of prostate cancer. Furthermore, we isolated tumor stem population from PC3mm, and examined the effect of BMP7 on these cells. BMP7 indeed induced p38, NDRG1 and p21 in the prostate tumor stem cells. BMP7 also significantly suppressed sphere forming ability of the tumor stem cells followed by activation of NDRG1 and this effect was reversed by withdrawal of BMP7. These results suggest that the BMP7-NDRG1axis plays a critical role in the regulation of dormancy and recurrence of prostate tumor cells in bone metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5762.