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Whether estrogen receptor beta (ERβ/ESR2) is a pro- or anti-oncogenic protein in breast cancer has been controversial. ERβ levels are generally high in triple-negative breast cancer (TNBC). Reports including the Cancer Genome Atlas (TCGA) show that about 80% of TNBC express mutant p53 and it is a major driver of these cancers. We tested the hypothesis that WT versus mutant status of p53 will have an important role in determining the duality of ERβ functions. We showed that ERβ directly binds to TP53 in human breast cancer cells. Using glutathione-S-transferase (GST)-pull down and co-imunoprecipitation assays, we have delineated the DNA binding domain (DBD) along with the hinge domain of ERβ and the C-terminal regulatory domain of p53 to be essential for the interaction. The highly sensitive in situ proximity ligation assay (PLA) showed that ERβ is capable of interacting with both wild type (WT) and mutant p53 in breast cancer cells and tumor tissues. ERβ and p53 antibodies validated for specificity were used for all experiments. Combination of proliferation, cell cycle, and apoptosis assays, RNAi technology, quantitative ChIP (qChIP), and real-time PCR (qRT-PCR) showed that ERβ is pro-proliferative in the context of WTp53, whereas it is anti-proliferative in the context of mutant p53 in multiple breast cancer cell lines. The p53-dependent diametrically opposite functions of ERβ were recapitulated in isogenic MDA-MB-231 TNBC cells (generated by CRISPR) that differ only in the presence of WT versus mutant p53. A major gain-of-function of mutant p53 is its ability to bind and inactivate tumor suppressor p73. We show that ERβ binds and sequesters mutant p53 from mutant p53−p73 complex leading to reactivation of p73. Consistent with these observations, immunohistochemistry (IHC) in TNBC patient tumor tissue microarray (TMA) showed that patients with tumors expressing mutant p53 along with high levels of ERβ were of smaller size and lower stage. Complementing these findings, our analysis of the subgroup of the basal-like/TNBC tumors expressing mutant p53 (but not WTp53) in the METABRIC dataset showed that patients with tumors expressing higher levels of ERβ RNA (ESR2) had significantly better breast cancer-specific survival. The finding that ERβ–mutant p53 combination prognosticates survival in TNBC is important as to date there are no effective prognostic markers for TNBC and suggest the potential for using ERβ−mutant p53 combination in stratification of TNBC into therapeutically actionable subgroups.Furthermore, our findings provide a mechanistic explanation for the functional duality of ERβ and the controversy over its pro-versus anti-tumorigenic role.Surprisingly, Tamoxifen (Tam) increased ERβ-mut-p53 interaction in TNBC cells leading to increased transcription of anti-proliferation genes and knockdown experiments showed that the effect on transcription was dependent on both p73 and ERβ. Importantly, when combined with doxorubicin (Adriamycin) Tam decreased several fold the IC50 of doxorubicin resulting in increased apoptosis. The combination was more effective in inhibiting TNBC xenograft tumor growth in vivo compared to either treatment alone. Significant clinical implications of these findings include the potential for treating patients with doxorubicin at much lower dose than what is currently used in the management of TNBC, thereby reducing its major cumulative dose side effects. Importantly, although at present Tam is not indicated for TNBC, our data suggest the possibility for repurposing Tam therapy alone or in combination with chemotherapy to treat TNBC stratified based on ERβ and p53 status. If validated in a clinical trial, our findings could lead to a therapy that is fundamentally better in terms of effectiveness, cost and time needed to reach the TNBC patients. Citation Format: Gokul M Das, Utpal K Mukhopadhyay, Chetan C Oturkar, Christina Adams, Nadi Wickramasekera, Sanjay Bansal, Rajesh Medisetty, Austin Miller, Wendy M Swetzig, Laxmi Silwal-Pandit, Anne-Lise Borresen-Dale, Creighton J Creighton, Jun Hyoung Park, Santhi D Konduri, Alka Mukhopadhyay, Alexander Caradori, Benny Abraham Kaipparettu. Exploiting p53-dependent functional duality of estrogen receptor-beta to repurpose tamoxifen for triple negative breast cancer therapy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-10-03.

Whether estrogen receptor beta (ERβ) is a pro- or anti-oncogenic protein in breast cancer has been controversial. ERβ levels are high in ERα negative cancers including triple-negative breast cancer (TNBCs). Recent reports including the Cancer Genome Atlas (TCGA) show that about 80% of TNBC express mutant p53 (mut-p53) and it is the most predominant driver in these cancers. We tested the hypothesis that p53 status in breast cancer will have an important role in determining the duality of ERβ functions. We have shown that ERβ directly binds to p53 in human breast cancer cells. Using glutathione-S-transferase (GST)-pull down and co-imunoprecipitation assays, we have delineated the domains of both proteins that are required for the ERβ-p53 interaction. The DNA binding domain (DBD) along with the hinge domain of ERβ and the C-terminal regulatory domain of p53 are essential for the interaction. Using the highly sensitive proximity ligation assay (PLA), we show ERβ-p53 interaction in situ in breast cancer cells expressing either wild type (wt)- or mut-p53. ERβ and p53 antibodies validated for specificity were used. In multiple cell lines, a combination of proliferation and apoptosis assays, RNAi technology, quantitative chromatin immunoprecipitation (qChIP), and quantitative real-time PCR (qRT-PCR) showed that ERβ is pro-proliferative in the context of wt-p53, whereas it is anti-proliferative in the context of mut-p53. The results were recapitulated in isogenic MDA-MB-231 TNBC cells (generated by CRISPR technology) that differ only in the presence of wt-versus mut-p53. ERβ binds and sequesters mut-p53 from mut-p53−p73 complex leading to reactivation of tumor suppressor p73. Consistent with these data, combination of immunohistochemistry (IHC) and PLA in TNBC patient tumor tissue microarray (TMA) showed that patients with tumors expressing wt-p53/ high ERβ had worse prognosis, both in terms of overall survival (OS) and progression-free survival (PFS). On the contrary, tumors expressing mut-p53/ high ERβ were of smaller size and stage. These findings were complemented by data from our analysis of the mut-p53 subgroup of the basal-like tumors in the METABRIC dataset which showed that patients with tumors expressing higher levels of ERβ RNA (ESR2) had better prognosis. Surprisingly, 4-Hyroxy Tamoxifen (Tam) increased ERβ-mut-p53 interaction in TNBC cells and combination of doxorubicin (Adriamycin) and Tam decreased the IC50 of doxorubicin by 10 fold leading to increased apoptosis. These data have significant clinical implications in targeting ERβ and mutant p53 signaling pathways for therapeutic purposes especially in ERα negative cancers such as TNBC. Importantly, although at present Tam is not standard of care for TNBC, our data suggest a possibility for repurposing Tam therapy alone or in combination with chemotherapy to treat TNBC stratified based on p53 status. Citation Format: Gokul M. Das, Utpal K. Mukhopadhyay, Christina Adams, Nadi Wickramasekera, Rajesh Medisetty, Sanjay Bansal, Laxmi Silwal-Pandit, Anne-Lise Borresen-Dale, Austin Miller, Wendy M. Sweizig, Angela Omilian, Wiam Bshara, Alka Mukhopadhyay. p53 status as a determinant of functional duality of estrogen receptor beta in breast cancer: Therapeutic implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3748.

Deregulation of apoptosis is central to cancer progression and a major obstacle to effective treatment. The Bcl-2 gene family members play important roles in the regulation of apoptosis and are frequently altered in cancers. One such member is Bcl-2-related Ovarian Killer (BOK), which is a pro-apoptotic protein. Despite its critical role in apoptosis, the regulation of BOK expression is poorly understood in cancers. Here, we discovered that miR-296-5p, regulates BOK expression by binding to its 3’UTR in breast cancers. Furthermore, we show that depletion of BOK by either miR-296-5p or siRNA against BOK protected breast cancer cells from undergoing paclitaxel-induced apoptosis. Interestingly, miR-296-5p also regulates the expression of Mcl-1, which is an anti-apoptotic protein and is highly expressed in breast cancers. Our results reveal that Mcl-1 is important for suppression of BOK function as ectopic BOK expression induced Mcl-1, while silencing of BOK resulted in reduced Mcl-1 levels in breast cancer cells. In addition, we show that specific silencing of Mcl-1 reduced the long-term growth of breast cancer cells, whereas BOK inhibition didn’t have any effect on the growth of breast cancer cells. Surprisingly, silencing of both Mcl-1 and BOK rescued the effect of Mcl-1 silencing on breast cancer cell growth, suggesting that BOK is important for attenuating cell growth in the absence of Mcl-1, and also showing a tight feedback regulatory loop between BOK and Mcl-1 in breast cancer cells. Furthermore, we demonstrated that BOK protein level is regulated post-translationally by GSK3α and to some extent GSK3β as GSK3 inhibitor (CHIR99021) or silencing of GSK3 significantly increased BOK protein levels in breast cancer cells. Notably, we found that Mcl-1 interacts with GSK3α/β and silencing of Mcl-1 using siRNA significantly attenuated endogenous GSK3α/β levels in breast cancer cells. Taken together, our results suggest that fine tuning (either post-transcriptionally by miR-296-5p or post-translationally by GSK3) of the levels of pro-apoptotic protein BOK and anti-apoptotic protein Mcl-1 decide the fate of cancer cells to either undergo Apoptosis or proliferation. Citation Format: Benjamin Chidi Onyeagucha, Panneerdoss Subbarayalu, Subapriya Rajamanickam, Nourhan Abdelfattah, Santosh Timilsina, Rosa M. Guzman, Carla Zeballos, Vijay Eedunuri, Sanjay Bansal, Hima Bansal, Tabrez A. Mohammad, Yidong Chen, Manjeet K. Rao. Novel regulatory mechanisms for Bcl2-related Ovarian Killer (BOK) expression in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2336. doi:10.1158/1538-7445.AM2017-2336

Microtubules are highly dynamic components of the cytoskeleton that play an important role in a wide range of cellular processes including cell division, cell motility and intracellular transport. Increasing evidence suggests that alterations in microtubule dynamics are critical for cancer growth and metastasis. Microtubule associated proteins (MAPs) regulate microtubule dynamics and consequently can affect sensitivity of cancer cells to microtubule targeting drugs. We discovered a novel microtubule associated protein "Matrin 3 (MATR3)" that is known to bind to RNA and play a critical role in RNA transport and RNA stabilization. Immunofluorescence analyses revealed that although MATR3 is predominantly a nuclear protein, it translocates to the cytoplasm and interacts with microtubules when breast cancer cells are treated with paclitaxel. We show that MATR3 associates with stabilized microtubules and it mediates microtubule polymerization in a taxol-dependent manner. Interestingly, our results reveal that MATR3 acts as a effective tumor suppressor as it inhibits breast cancer colony formation, migration and invasion of breast cancer cells in addition to suppressing breast tumor growth in vivo. Analysis of breast cancer samples showed a significantly lower expression of MATR3 when compared to normal adjacent tissues. Our results indicate the possibility that MATR3 mediates its tumor suppressor function by binding and regulating proteins that are known to affect microtubule dynamics. We believe that nuclear-cytoplasmic shuttling of MATR3 is critical for stability of key proteins that might regulate paclitaxel-dependent microtubule dynamics and subsequently cellular effects in cancer cells. Citation Format: Panneerdoss Subbarayalu, Subapriya Rajamanickam, Suryavathi Viswanadhapalli, Nicholas Dybdal-Hargreaves, Santosh Timilsina, Sanjay Bansal, Hima Bansal, Tabrez Mohammad, Yidong Chen, John C Herr, Susan L Mooberry, Manjeet K Rao. Matrin 3: A novel micro-tubule associated RNA binding protein that acts as a potent tumor suppressor [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-05-09.

Microtubule-targeting drugs are widely used as cancer chemotherapeutic agents and they have been shown to inhibit mitotic progression and interphase signaling. Despite their use as a first line treatment for cancer, many patients develop resistance to microtubule-targeting drugs leading to early relapse and shorter survival. Moreover, the quality of life for patients who do survive is often substantially reduced due to the toxicity associated with these drugs. Therefore, identification of new factors that determine the effectiveness of microtubule-targeting agents will not only facilitate a better understanding of the mechanisms of acquired drug resistance but will also be amenable to therapeutic interventions. We discovered a novel microtubule associated protein “Matrin 3 (MATR3)” that is known to bind to RNA and play a critical role in RNA transport and RNA stabilization. Our results revealed that MATR3 acts as a potent tumor suppressor as it inhibits long-term growth, migration, invasion as well as tumor growth of triple negative breast cancer (TNBC) cells in vivo. We demonstrated that MATR3 overexpression induced cell cycle arrest and apoptosis in TNBC cells. Furthermore, analysis of breast cancer samples showed a significantly lower expression of MATR3 when compared to normal adjacent tissues. Importantly, our RNA immunoprecipitation (RIP)-seq analysis showed that MATR3 controls expression of several mitotic spindle organizing proteins by binding to their RNA. Interestingly, we found that these MATR3 regulated proteins were highly altered in breast cancer patients. In conclusion, we identified a novel RNA-binding protein that inhibits breast cancer growth and progression suppressor by regulating microtubule dynamics. Citation Format: Panneerdoss Subbarayalu, Subapriya Rajamanickam, Suryavathi Viswanadhapalli, Benjamin C. Onyeagucha, Vijay K. Eedunuri, Nicholas Dybdal-Hargreaves, Santosh Timilsina, Hima Bansal, Sanjay Bansal, Tabrez Mohammad, Yidong Chen, John C. Herr, Susan L. Mooberry, Manjeet K. Rao. A novel microtubule associated RNA binding protein matrin 3 act as a tumor suppressor by regulating mitotic spindle organizing proteins in triple negative breast cancers. [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 3658.

Background: Histone deacetylase (HDAC) inhibitors, such as vorinostat, have been of interest in adult and pediatric cancers. Preclinical in vitro data has shown an effect in many different cancer cell types, but response has not correlated well in xenograft models. A concern is the effect of the HDAC inhibitors being compensated by autophagy. Autophagy is induced by multiple anticancer agents, as a tumor survival-promoting mechanism. Hydroxychloroquine, an autophagy inhibitor, treatment leads to the accumulation of autophagosomes, and accelerates tumor cell death. The hypothesis of this study is that by combining a HDAC inhibitor with hydroxychloroquine, there will be an improved anti-tumor response compared to HDAC inhibitor alone in pediatric cancer cell lines. Objectives: To determine if there is an improved efficacy of vorinostat when combined with hydroxychloroquine in pediatric solid tumor cell lines. Methods: Three pediatric cancer cell lines: Hepatoblastoma (Hep293TT), Ewing's Sarcoma (SKES1), and Medulloblastoma (D283) were treated with different concentrations of vorinostat or hydroxychloroquine, and cell viability was measured by CellTiter Glo assay. Dose-response curves were plotted to determine IC50 (inhibitory concentration 50%). The expression of proliferation and autophagy markers was determined by Western blot analysis. Results: The IC50 of vorinostat and hydroxychloroquine was in the rage of 0.5-1.25μM and 25-50μM, respectively. To evaluate the functional consequences, cells were treated with vorinostat and hydroxychloroquine alone and in combination for 72 hours. The combination treatment resulted in a dramatic decrease in cell viability, with these two drugs cooperating in a dose-dependent manner. Overall, in all three cell lines there was an improved response when vorinostat was combined with hydroxychloroquine. LC3, a mammalian homolog of yeast Atg8, is a reliable marker of autophagosomes, and tracking the conversion of LC3-I to LC3-II is indicative of autophagy blockade. Hydroxychloroquine treatment resulted in profound conversion of LC3-I to LC3-II, and this effect was greater in cells treated with both vorinostat and hydroxychloroquine alone. Therefore, hydroxychloroquine blocked autophagic flux, which was more pronounced in the setting of autophagy induction by vorinostat. Finally, exposure to the combination of vorinostat and hydroxychloroquine resulted in decreased levels of phospho-ERK compared to single-agent treatment, indicating reduced cell proliferation. Taken together, our data demonstrate that when autophagy was inhibited by hydroxychloroquine in the presence of vorinostat, autophagosomes accumulated and cell viability/proliferation reduced. Conclusion: Preclinical studies in the different pediatric cancer cell lines show a clear and improved response when vorinostat is combined with hydroxychloroquine compared to vorinostat alone. The increased LC3-II/I ratio indicative of efficient autophagy inhibition while the decrease in the phospho-ERK signifies a decrease in proliferation. Furthermore the IC50 concentration of vorinostat, about 200ng/ml, necessary for effect is well within the achievable clinical range of 150-500ng/ml. The next step would be to validate these results in a xenograft model with one of the pediatric cancer tumors, and then hopefully continue forward to build a phase 1 clinical trial in recurrent pediatric solid tumors. This combination of drugs has yet to be explored in pediatrics but has the potential to enhance the effect of an established chemotherapy agent with a well-known and easily accessible non-cancer drug. Citation Format: Abigail Joy Cruz, Hima Bansal, Gail Tomlinson, Sanjay Bansal, Steven Weitman. Enhancement of histone deacetylase inhibitor's efficacy with hydroxychloroquine via autophagy inhibition in pediatric tumor cell lines. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B36.

Estrogen receptor α (ERα) and tumor suppressor protein p53 exert opposing effects on cellular proliferation. As a transcriptional regulator, p53 is capable of activating or repressing various target genes. We have previously reported that ERα binds directly to p53, leading to down-regulation of transcriptional activation by p53. In addition to transcriptional activation, transcriptional repression of a subset of target genes by p53 plays important roles in diverse biological processes, such as apoptosis. Here, we report that ERα inhibits p53-mediated transcriptional repression. Chromatin immunoprecipitation assays reveal that ERα interacts in vivo with p53 bound to promoters of Survivin and multidrug resistance gene 1, both targets for transcriptional repression by p53. ERα binding to p53 leads to inhibition of p53-mediated transcriptional regulation of these genes in human cancer cells. Transcriptional derepression of Survivin by ERα is dependent on the p53-binding site on the Survivin promoter, consistent with our observation that p53 is necessary for ERα to access the promoters. Importantly, mutagenic conversion of this site to an activation element enabled ERα to repress p53-mediated transcriptional activation. Further, RNA interference–mediated knockdown of ERα resulted in reduced Survivin expression and enhanced the propensity of MCF-7 cells to undergo apoptosis in response to staurosporine treatment, an effect that was blocked by exogenous expression of Survivin. These results unravel a novel mechanism by which ERα opposes p53-mediated apoptosis in breast cancer cells. The findings could have translational implications in developing new therapeutic and prevention strategies against breast cancer. [Cancer Res 2007;67(16):7746–55]

While the importance of estrogen receptor alpha (ERα) in breast cancer is well documented, role of estrogen receptor beta (ERβ) still remains elusive. Whether ERβ is an oncogenic or a tumor suppressor protein in breast cancer remains controversial. ERβ levels are high in ERα negative cancers including triple-negative breast cancer (TNBCs). Recent reports including the Cancer Genome Atlas (TCGA) show that about 80% of TNBC express mutant p53 (mut-p53) and it is the most predominant driver mutant in these cancers. Importantly, in addition to losing tumor suppressor activity, mut-p53 gains oncogenic functions, and therefore, has a major role in driving the cancer process. We tested the hypothesis that p53 status in breast cancer will have an important role in determining function of ERβ. We report that ERβ directly binds to p53 in human breast cancer cells. Using glutathione-S-transferase (GST)-pull down and co-imunoprecipitation assays, we have delineated the domains of both proteins that are required for the ERβ-p53 interaction. The DNA binding domain (DBD) along with the hinge domain of ERβ and the C-terminal regulatory domain of p53 are essential for the interaction. Using the highly sensitive proximity ligation assay (PLA), we show ERβ-p53 interaction in situ in breast cancer cells expressing either wt- or mut-p53. Surprisingly, we found that ERβ has opposite functions depending on the wt/mut status of p53. A combination of proliferation and apoptosis assays, RNAi technology, quantitative chromatin immunoprecipitation (qChIP), and quantitative real-time PCR (qRT-PCR) showed that in the context of the wt-p53, ERβ is pro-proliferative, whereas in the context of mut-p53, ERβ is anti-proliferative. ERβ binds wt-p53 and inhibits its transcriptional function. On the other hand, ERβ binds and sequesters mut-p53 from mut-p53−p73 complex leading to reactivation of tumor suppressor p73. Consistent with findings in cell culture models, combination of immunohistochemistry (IHC) and PLA in TNBC patient tissue microarray (TMA) representing 130 tumors followed by correlative analysis of linked patient tumor characteristics obtained from RPCI clinical data network showed that p53 status is an important determinant of ERβ function. For example, ERβ along with mut-p53 has negative effect on tumor growth, whereas ERβ in the wt-p53 context has opposite effect. Our finding that ERβ elicits opposite functions dependent on p53 status provides an important insight into the mechanism underlying the context-dependent function of ERβ and provides an explanation to the controversial reports on pro- versus anti-tumorigenic role of ERβ. These data would have significant clinical implications in targeting ERβ and mutant p53 signaling pathways for diagnostic, prognostic, and therapeutic purposes especially in ERα negative cancers such as triple negative breast cancer. Note: This abstract was not presented at the meeting. Citation Format: Gokul M. Das, Utpal K. Mukhopadhyay, Sanjay Bansal, Nadi Wickramasekera, Rajesh Medisetty, Wendy M. Swetzig, Austin Miller, Jianmin Wang, Chetan Oturkar, Alka Mukhopadhyay, Santhi Konduri. p53 status as a determinant of estrogen receptor beta function in breast 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 3465. doi:10.1158/1538-7445.AM2015-3465

Background: Hepatoblastoma (HB) is the most common primary, malignant liver tumor in children, occurring primarily in children under the age of 4 years. Although chemotherapy in conjunction with surgical resection has improved the prognosis for patients with HB, disease-free survival rates remain less than 50% for patients with advanced, metastatic or relapsed disease, highlighting the need for novel treatments. Additional subsets of tumors with a worse prognosis include those with histologic features of hepatocellular carcinoma (HCC) and tumors which develop in children older than 4 years. Nuclear factor erythroid-2–related factor 2 (NFE2L2, also known as Nrf2), a key transcriptional regulator for the cellular response to oxidative stress in normal cells, has been shown to contribute to malignant phenotypes of cancers. Recently, activating mutations in NFE2L2 have been reported in HCC and also in HB by our research team (Trevino et al1). These activating mutations of NFE2L2 and/or high expression of NFE2L2 have been observed notably in several pediatric liver tumors with high-risk features associated with a poor outcome. We hypothesize that NFE2L2 activation/overexpression may contribute to an aggressive tumor phenotype, and investigated its role in HB cell proliferation. Methods: Hep293TT cells, previously established using primary tumor tissues from a highly aggressive hepatoblastoma from a 5-year-old Caucasian female, were transfected with NFE2L2 small hairpin RNAs (shRNAs) to determine the effect of NFE2L2 suppression on cell proliferation and to identify potential NFE2L2-regulated targets. Analyses of Hep293TT cell proliferation and cell cycle kinetics were conducted using cell viability assays (CellTiter-Glo Luminescence), propidium iodide staining and FACS analysis. A comprehensive transcriptome analyses of Hep293TT cells was carried out by performing RNA sequencing (RNA-seq) using an Illumina HiSeq 2000. Results: RNA-seq analysis was performed on a set of pediatric cancer cell lines including Hep293TT, as well as reference liver cell lines HepG2 and Hep3B. After normalization, the analysis revealed high expression of NFE2L2 in Hep293TT cells with fold change of 2.01 relative to the mean expression level from the pediatric cancer cell line panel (14 cell lines). Compared to other liver cancer cell lines, fold change of 1.28 and 1.5 were measured from Hep3B and HepG2, respectively. Differential expression between NFE2L2 knockdown and parental cell will further demonstrate the molecular and functional influence of NFE2L2. Furthermore, knockdown of NFE2L2 inhibited cell proliferation in Hep293TT cells by inducing cycle arrest. Conclusions: Our results suggest that NFE2L2 may contribute to cell growth and proliferation in hepatoblastoma. Further studies are warranted to determine if NFE2L2 status can be used to stratify highly aggressive pediatric liver tumors and establish NFE2L2 as a potentially novel target for the treatment of HBs. 1Trevino et al, AACR Annual Meeting, 2012, Abstract #4592 This work was supported by grants from the Cancer Prevention and Research Institute of Texas RP101195 and NIH R21 CA122565. Citation Format: Hima Bansal, Tina Chen, Zhao Lai, Donald Parsons, Lisa Trevino, Dolores Lopez-Terrada, Milton Finegold, Dinesh Rakheja, Sarah Comerford, Robert Hammer, Bradley Pollock, Jaclyn Hung, Sanjay Bansal, Yidong Chen, Gail Tomlinson. Silencing of Nuclear factor (erythroid-derived 2)-like 2 (NFE2L2) impairs growth and proliferation of hepatoblastoma cells. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A17.

Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis that may have promise as an anticancer agent due to its selectivity toward tumor cells. However, resistance to TRAIL occurs frequently and is primarily due to defects in apoptosis signaling or activation of survival pathways. Sunitinib is an inhibitor of multiple receptor tyrosine kinases that promote cell survival and drug resistance. We hypothesized that disrupting pro-survival signaling cascades with suntinib would circumvent this mechanism of resistance and strongly enhance TRAIL-mediated apoptosis. We tested this hypothesis by investigating whether inhibition of survival signaling pathways with sunitinib could augment the anticancer activity of TRAIL in preclinical models of colon cancer. Sunitinib significantly enhanced the anticancer activity of TRAIL in a panel of colon cancer cell lines as evidenced by increased caspase cleavage and DNA fragmentation. The TRAIL/sunitinib combination also promoted enhanced JNK activation and stimulated a decrease in the expression of XIAP, c-IAP2 and Bcl-xL, which correlated with apoptosis. siRNA-mediated knockdown of JNK reduced TRAIL/sunitinib-mediated apoptosis indicating that it is an important mediator of efficacy of this combination. We further investigated this combination regimen in HCT116 and HCT15 xenograft models treated with vehicle, TRAIL, sunitinib, or the combination. The TRAIL/sunitinib combination was very well tolerated and significantly reduced tumor burden in both models compared to either single agent treatment. The reduction in tumor volume correlated with increased apoptosis as measured by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and was associated with decreases in XIAP, c-IAP2 and Bcl-xL expression. Our collective results suggest that sunitinib enhances TRAIL-mediated apoptosis by increasing JNK activation and downregulating multiple anti-apoptotic proteins. These findings are the first report of the therapeutic potential of the combination of sunitinib and TRAIL in preclinical models of colon cancer. 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 669.