Role Of FOXA1 Research Articles

Abstract C076: New insights into the role of FOXA1- HNF4 axis in pancreatic cancer

Abstract Despite recent advances in the treatment of pancreatic adenocarcinoma (PDAC), the median survival remains <12 months. Patients typically present with late-stage disease and have limited treatment options. Therefore, there is an immediate need for the generation of new and innovative therapeutic targets. Little is known about the role of pioneer factors such as FOXA1 and their interaction partners in a PDAC context. Inspired by literature reports implicating FOXA1 and GATA5/GATA6 in regulating pancreatic cancer resistance and metastasis, our study hypothesised the possibility of an undiscovered nuclear receptor that works with FOXA1 (similar to Estrogen receptor in breast cancer). Using innovative ‘omic’ based approaches (RIME) developed in our laboratory 1 we discovered a nuclear receptor (NR) complex involving HNF4A and HNF4G in the classical sub-type of pancreatic cancer. Subsequently the interaction was independently validated in Whipple surgical biopsies from PDAC patients using ChIP-sequencing studies. Across multiple patient tumors (n=7) a binding overlap specifically between FOXA1 and HNF4G (3461 sites) was established. To investigate the therapeutic potential of HNF4G in the classical subtype of PDAC, we generated CRISPR deletions (KO) of HNF4G in the HPAF-II cells. HNF4G-KO cells were orthotopically implanted into the pancreas of NSG mice. A significant survival advantage of 12 days (<0.001) and reduced growth (<0.02) was observed in these mice compared to the controls. To better understand the impact of HNF4G-KO on gene expression, the orthotopic tumors were subjected to RNA-seq analyses. Gene set enrichment and pathway analysis revealed significantly down-regulated pathways to include EMT transition. TCGA data highlighted HNF4G amplification in 9% of PDAC patients with concomitant decreased progression free survival. These data point towards HNF4G being a therapeutically viable target for further exploration. Protein Arginine Methyl Transferase 1 (PRMT1) is a common interactor of both FOXA1 and HNF4G. Our study reveals a unique dependency of PRMT1 on the HNF4G-FOXA1 complex in PDAC biopsies. HNF4G-KO drastically reduces the chromatin binding of PRMT1, implicating them as functionally dependent. Treatment of HNF4G-KO cells with GSK3368715 (PRMT1 inhibitor) further sensitizes these cells and results in a significant survival advantage (10 days <0.02). We propose a model of HNF4G inhibition in combination with PRMT1 as a novel therapeutic opportunity to treat the classical sub-type of PDAC. Further, our study reveals a unique reliance of primary classical tumors on HNF4G. Although HNF4G appears to dominate FOXA1 functionality in the primary disease, FOXA1 remains instrumental in priming metastasis and enhancer reprograming. 1. Papachristou EK, Kishore K, Holding AN, Harvey K, Roumeliotis TI, Chilamakuri CSR, et al. A quantitative mass spectrometry-based approach to monitor the dynamics of endogenous chromatin-associated protein complexes. Nature Communications. 2018;9(1):2311. Citation Format: Shalini V. Rao, Lisa Young, Danya Cheeseman, Stephanie Mack, Jill Temple, Chandra Shekar Reddy Chilamakuri, Evangelia Papachristou, Catherina Pelicano, Amy Smith, Dominique-Laurent Couturier, Michael Gill, Duncan Jodrell Jodrell, Alasdair Russell, Igor Chernukhin, Jason Carroll. New insights into the role of FOXA1- HNF4 axis in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C076.

FOXA1在恶性肿瘤中的作用及其机制研究进展

FOXA1在恶性肿瘤中的作用及其机制研究进展

New insights into the role of FOXA1- HNF4 axis in Pancreatic Cancer

New insights into the role of FOXA1- HNF4 axis in Pancreatic Cancer

FOXA1 is associated with high tumor grade, myometrial invasion and lymph node invasion in endometrial endometrioid carcinoma.

FOXA1 expression has been demonstrated in several hormone-dependent cancers. However, data are limited concerning the role of FOXA1 in endometrial cancers. The present study aimed to investigate FOXA1 expression via the microarray technique in benign hyperplasia, endometrial intraepithelial neoplasia, and endometrial endometrioid carcinoma. We also aimed to determine whether there were any associations between FOXA1 expression, tumor grade, myometrial invasion and lymphatic invasion. Paraffin-embedded sections prepared from samples obtained from 114 patients who underwent surgical hysterectomy or curettage were analyzed. Data were retrieved from digitally-stored medical records. Tissue microarrays were prepared from formalin-fixed, paraffin-embedded tissue blocks. Full tumor sections were used for immunohistochemical analysis performed. Carcinomas with nuclear grade 3 had higher FOXA1 values than others, while grade 2 carcinomas also had higher FOXA1 values relative to grade 1 (p < 0.001). FOXA1 values of FIGO stage III carcinomas were significantly higher than others and stage II values were also significantly higher than stage I FOXA1 values (p < 0.001). Patients with myometrial and lymph node invasion had significantly higher FOXA1 values than others (p < 0.001 and p = 0.047, respectively). FOXA1 had 91.30% sensitivity, 63.60% specificity and 77.78% accuracy for predicting the presence of myometrial invasion with a cut-off value of 9. FOXA1 expression is higher in endometrial endometrioid carcinoma compared to benign endometrial hyperplasia or intraepithelial neoplasia. In patients with endometrial endometrioid carcinoma, high FOXA1 expression is associated with high tumor grade, myometrial and lymph node invasion. However, FOXA1 expression is not associated with lymphovascular or cervical invasion.

FOXA1 Expression by Immunohistochemistry in Carcinosarcomas of the Endometrium and Ovary/Fallopian Tube.

FOXA1, a transcription factor essential for the binding of other transcription factors on chromatin, is associated with hormone receptor-associated cancers, such as breast and endometrial cancer. It is also considered an antagonist of epithelial-to-mesenchymal transition (EMT). In endometrial cancer, FOXA1 is considered a tumor suppressor; in carcinosarcoma, one of the most aggressive and rare subtypes of endometrial cancer, thought to be derived through an EMT mechanism, FOXA1 has not been studied. Thus, the aim of this study was to investigate the possible expression of FOXA1 in carcinosarcomas, and its correlation with clinicopathologic factors. This was a retrospective study of 31 patients diagnosed with carcinosarcomas of the uterus or the adnexa. Histologic and clinical factors were correlated with the immunohistochemical expression of FOXA1. FOXA1 was expressed by 38.7% of the carcinomatous components and 16.1% of the sarcomatous components. FOXA1-positive sarcomatous components were seen only with positive carcinomatous components (P=0.004). FOXA1 expression was not associated with age, primary tumor site, stage, metastases, overall survival, or tumor relapse. FOXA1 expression in the carcinomatous component was associated with an absence of lymphovascular invasion or the presence of heterologous components. FOXA1 expression in the sarcomatous component was associated with rhabdomyosarcoma, rather than the chondrosarcoma heterologous component. Carcinosarcomas harbor FOXA1 expression, although it is in their carcinomatous rather than sarcomatous components, suggesting a possible role of FOXA1 in the EMT of carcinosarcomas. FOXA1 shows no prognostic significance in this tumor group.

Abstract PS4-12: Potential value of FOXA1 expression as genomic predictor of response to docetaxel and carboplatin neoadjuvant (NA) chemotherapy in patients with triple negative breast cancer (TNBC)

Abstract Background: TNBC is a particularly aggressive subtype with high recurrence rates and poor long-term survival. Anthracycline-free regimens based on docetaxel plus carboplatin (TCb) achieve around 50-55% pathologic complete responses (pCR) which correlates with better survival outcomes. Identification of genomic predictors of response is key to individualize therapies for this heterogeneous breast cancer (BC) subgroup. FOXA1 is a pioneer factor for androgen (AR) and estrogen receptors (ER). Its under-expression has been linked to cancer stemness in TNBC and better prognosis in ER-positive BC. The differential expression of AR, FOXA1 and BRCA1 is associated with sensitivity to chemotherapy in TNBC patients. Data support the plasticity role of FOXA1 driving basal to luminal BC cells by inducing luminal genes but also by repressing the basal phenotype and thus, tumor aggressiveness, although its role as a biomarker in TNBC is still controversial. We have analyzed RNAseq data of FOXA1 and AR in a series of TNBC tumors homogenously treated with neoadjuvant TCb to study their correlation with survival and response. Methodology: 300 TNBC patients have been included in a multicenter, prospective, non-randomized trial aimed to identify predictors of response to TCb in the neoadjuvant setting (NCT01560663). 278/300 patients were evaluable. PAM50 subtypes (NanoString nCounter) and RNAseq (HiSeq2500) were analyzed for those patients with both available samples and evaluable pathological response (n=208). Correlations were studied by the Kendall method. Linear or logistic regressions were adjusted for univariant analysis depending on numerical or categorical response variables, respectively. Results: FOXA1 and AR were differentially expressed between basal and non-basal tumor subtypes (PAM50), being overexpressed in non-basal (p&amp;lt;0.001 for both FOXA1 and AR). FOXA1 and AR were significantly overexpressed in non-basal subtypes from the Vanderbilt TNBC-type classification (36/208) as well. Both genes expression showed a significant correlation with several luminal marker genes. AR was significantly associated with ANXA9 (p=0,008), GATA3 (p=0,00002) and XBP1 (p&amp;lt;0.001), while FOXA1 expression was positively associated with ANXA9 (p=0,03), ESR1 (p&amp;lt;0.001),GATA3 (p&amp;lt;0.001) and XBP1 (p&amp;lt;0.001). In terms of response, significant differences in FOXA1 expression levels were found between responders and non-responders by Residual Disease Burden (RCB) classification taking response as binary variable of PCR vs RCB-I/II/III (p=0.003) as well as pCR/RCB-I vs RCB-II/III (p=0.008) with responders showing a lower FOXA1 expression level. The same analysis for AR expression did not show statistically significant differences. Within the PAM50 basal subgroup, neither AR nor FOXA1 individual gene expression (172/208) showed association with response. For survival, with a median follow-up of 36 months, neither FOXA1 nor AR showed significant impact on EFS or OS per univariate Cox regression analysis (EFS: HRFOXA1 1.076; p=0.307; HRAR 1.156, p=0.084 and OS: HRFOXA11.120, p=0.140; HRAR 1.152, p=0.121). Conclusions: Within our cohort, FOXA1 expression is a better predictor of response to NA chemotherapy than AR. FOXA1 is overexpressed in TNBC tumors who do not achieve pCR with neoadjuvant TCb chemotherapy, correlating with non-basal tumor subtypes. This suggests that measuring FOXA1 expression levels in patients with TNBC could be useful to predict response to TCb regimen enabling physicians to select more effective alternative NA schemes in this population. Citation Format: Sara López-Tarruella, María del Monte-Millán, Enrique Alvarez, Yolanda Jerez, José Ángel García Saenz, Isabel Echavarria, Fernando Moreno, Ivan Márquez-Rodas, Coralia Bueno-Muiño, Blanca Herrero, Maria Cebollero, Rocío Ramos-Medina, Nerea Lobato, Ricardo González del Val, Maria Isabel Palomero, Santiago Lizarraga, Uriel Bohn, Ana Isabel Ballesteros, Patricia Rincón, Tatiana Massarrah, Inmaculada Ocaña, Lucía Villarejo, Charles M Perou, Miguel Martin. Potential value of FOXA1 expression as genomic predictor of response to docetaxel and carboplatin neoadjuvant (NA) chemotherapy in patients with triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-12.

Abstract 3437: Cistromically-dominant class2 mutants of FOXA1 de-repress WNT signaling to drive prostate cancer metastasis

Abstract Forkhead box A1 (FOXA1) is a pioneer transcription factor that is essential for the normal development of several endoderm-derived organs, including the prostate gland. FOXA1 independently binds to and de-compacts condensed chromatin to reveal the binding sites of partnering nuclear hormone receptors. In prostate luminal epithelial cells, FOXA1 delimits tissue-specific enhancers that are transcriptionally activated by the androgen receptor (AR). Thus, it is intriguing that FOXA1 is frequently mutated in hormone-receptor-driven prostate, breast, bladder and salivary-gland tumors. In a recent study, leveraging an aggregate cohort of 1,546 prostate cancers, we classified FOXA1 alterations into three novel structural classes that diverge in clinical incidence and genetic co-alteration profiles, with a collective prevalence of 35%. Class1 mutations comprise of missense and in-frame indels within the Wing2 secondary structure and originate in early prostate cancer without other driver alterations in genes such as ETS or SPOP. Contrarily, class2 mutations comprise of frameshift mutations which truncate the C-terminal end of the protein and are clonally acquired in the metastatic disease. Finally, class-3 genomic rearrangements comprise of duplications and translocations that reposition a conserved enhancer element to drive overexpression of FOXA1 or other oncogenes. Here, we mechanistically expand upon the neomorphic class2 mutants and show that they centrally enable prostate cancer metastasis. Remarkably, we found truncated class2 mutants to entirely displace their wild-type counterpart from the chromatin - a characteristic we termed as “cistromic dominance” - due to higher DNA affinity. Through proteomic assays, we identified the C-terminal regulatory domain of wild-type FOXA1 to interact with and recruit a bonafide transcriptional WNT-repressor called TLE3 to the chromatin. Notably, class2 mutants had lost this interaction and dominantly displace TLE3 from chromatin to de-repress WNT signaling. Accordingly, we found CRISPR-engineered prostate cancer cells harboring class2 FOXA1 mutations to show higher invasive and metastatic ability both in vitro and in vivo. Also, the increase in the metastatic ability of class2-mutant cells was completely reversed upon treatment with a WNT inhibitor. Overall, our study reaffirms the central role of FOXA1 in mediating AR-driven oncogenesis and provides mechanistic insights into how distinct classes of FOXA1 alterations drive initiation or metastatic progression of prostate cancer. These results reveal a novel FOXA1-TLE3-WNT signaling axis and rationalize its therapeutic co-inhibition in advanced prostate cancer. Citation Format: Abhijit Parolia, Marcin Cieslik, Sandra Carson, Shih-Chun Chu, Lanbo Xiao, Takahiro Ouchi, Mustapha Jaber, Yuping Zhang, Pankaj Vats, Xuhong Cao, Arul Chinnaiyan. Cistromically-dominant class2 mutants of FOXA1 de-repress WNT signaling to drive prostate cancer metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3437.

Abstract 923: FOXA1 chromatin binding is regulated by LSD1-mediated methylation in prostate cancer cells

Abstract Androgen receptor (AR) plays a critical function in driving prostate cancer (PCa) development and androgen deprivation therapy (ADT) is standard of care in treating PCa patients. Although patients generally response well, the cancer eventually relapse and progress to the castration-resistant stage of PCa (called CRPC), which can be further treated with more aggressive ADT. However, the tumors still develop resistance within a year and restored AR activity is found in the majority of relapsed tumors, indicating a pressing need for developing novel AR-targeted therapies. In PCa cells, AR activity is dependent on the chromatin binding of the pioneer factor, FOXA1. In particular, FOXA1 recognizes the H3K4me1,2 marked enhancers and its binding opens the chromatin for subsequent AR recruitment. Although the role of FOXA1 on AR signaling has been intensively studied, the mechanisms regulating FOXA1 binding to chromatin remain elusive. LSD1/KDM1A was initially identified as a transcription repressor through demethylation of H3K4me1,2. In contrast to its well-established corepressor function, LSD1 has been found to coactivate several transcription factors including AR. This AR coactivator function has been attributed to the phosphorylation of histone 3 on threonine 6 and threonine 11, which may switch LSD1 substrate specificity from H3K4me1,2 to the repressive mark H3K9me1,2. However, we have recently reported that the H3K4 demethylase activity of LSD1 persists at AR-regulated enhancer sites, including sites marked by H3T6ph, arguing against the coactivator function of LSD1 being generally controlled by this mechanism. Importantly, we have shown in a recent study that LSD1 associates with FOXA1 and active enhancer markers, and that LSD1 silencing impaired FOXA1 binding at AR-regulated enhancers, suggesting a role of LSD1 in regulating enhancer availability to AR. In the current study, we found that LSD1 inhibition globally disrupts FOXA1 chromatin binding prior to androgen stimulation and thus impairs further AR recruitment, resulting in the global inhibition of AR transcriptome. Using immunoprecipitation and mass-spectrometry assays, we discovered that LSD1 maintains FOXA1 binding by demethylating lysine K270, adjacent to its DNA binding domain. We further found that the expression of a methylation-deficient FOXA1 mutant (K270R) stabilizes AR chromatin binding and allows PCa cells resistant to enzalutamide treatment. Using a clinical tested LSD1 inhibitor, we have shown in vivothat LSD1 inhibition dramatically decreases PCa growth alone and in synergy with enzalutamide in AR/AR variants-positive CRPC models. These findings provide fundamental new insights into FOXA1, LSD1, and mechanisms that regulate the activity of AR in PCa cells. As LSD1 inhibitors are moving into the clinic, these new insights into LSD1 function strongly support LSD1 as a therapeutic target in CRPC. Citation Format: Shuai Gao, Sujun Chen, Dong Han, Wanting Han, Jude N. Owiredu, Steven P. Balk, Housheng H. He, Changmeng Cai. FOXA1 chromatin binding is regulated by LSD1-mediated methylation in prostate cancer cells [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 923.

Abstract 4497: Distinct structural classes of activating FOXA1 alterations in prostate cancer progression

Abstract Forkhead box A1 (FOXA1) is a pioneer transcription factor that is essential for the normal development of several endoderm-derived organs, including the prostate gland. FOXA1 is frequently mutated in the hormone-receptor driven prostate, breast, bladder, and salivary gland tumors. In prostate luminal epithelial cells, wild-type FOXA1 delimits tissue-specific enhancers that are transcriptionally activated by AR, and extensively reprograms AR-activity in the transformed prostate epithelia. However, how FOXA1 alterations affect cancer development is unclear, with FOXA1 previously ascribed both tumor suppressive and oncogenic roles. In this study, we assemble an aggregate cohort of 1546 prostate cancers (PCa) and, for the first time, show that FOXA1 alterations fall into three distinct structural classes that diverge in clinical incidence, genetic co-alteration profiles, and oncogenic gain-of-functions. Notably, we find the three classes of FOXA1 alterations to collectively recur at a frequency of 35% in metastatic PCa. Class1 activating mutations originate in early PCa without concurrent ETS or SPOP alterations, selectively recur within the Wing2-region of the DNA-binding Forkhead domain (FKHD), confer enhanced chromatin mobility and binding frequency, and strongly trans-activate a luminal androgen receptor (AR) program of prostate oncogenesis. By contrast, class2 activating mutations are acquired in metastatic PCa, truncate the C-terminal regulatory domain of FOXA1, confer chromatin-binding dominance by increasing DNA affinity, and aberrantly activate the WNT/β-Catenin pathway to enable PCa metastasis. Finally, class3 genomic rearrangements are comprised of tandem duplications and translocations within the highly-syntenic FOXA1 locus. These structural variations amplify or reposition a conserved enhancer element, which we named FOXA1 Mastermind (FOXMIND), to drive overexpression of FOXA1 or other oncogenes, respectively. In summary, our study reaffirms the central role of FOXA1 in mediating AR-driven oncogenesis, and provides mechanistic insights into how different classes of FOXA1 alterations uniquely promote PCa initiation and/or metastatic progression. Furthermore, these results have direct implications in understanding the biology of other hormone-receptor driven cancers where similar FOXA1 alterations are found, and rationalize therapeutic co-targeting of oncogenic FOXA1-activity to extort more potent and durable disease remissions. Citation Format: Abhijit Parolia, Marcin Cieslik, Shih-Chun Chu, Lanbo Xiao, Takahiro Ouchi, Yuping Zhang, Xiaoju Wang, Pankaj Vats, Xuhong Cao, Fengyun Su, Rui Wang, Felix Feng, Yi-Mi Wu, Robert Lonigro, Dan R. Robinson, Arul M. Chinnaiyan. Distinct structural classes of activating FOXA1 alterations in prostate cancer progression [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 4497.

Abstract 976: FOXA1 promotes a luminal growth program in prostate cancer

Abstract FOXA1 is an important pioneering transcription factor for androgen receptor (AR), a key driver of prostate cancer. FOXA1 has emerged as one of the most recurrently mutated genes in primary and metastatic castrate resistant prostate cancer, with alterations frequencies ranging from 4-15% in various cohorts. Given the well-established role of FOXA1 in modulating AR function, we hypothesize that FOXA1 mutations contribute to prostate oncogenesis by altering the chromatin landscape, resulting in changes in the AR cistrome and AR transcriptional output. Here we use prostate organoids to show that acute FOXA1WT overexpression promotes lumen formation and drives luminal differentiation. Consistent with this, CRISPR/Cas9-mediated deletion of FOXA1 slows proliferation and organoids lacking FOXA1 fail to form lumens. Assays to evaluate the functionality of recurrent FOXA1 mutations (measured by cell viability, lumen formation, and lumen size) along with RNA-sequencing suggest there are distinct classes of FOXA1 mutations. The first are the gain-of-function mutations (such as FOXA1FE254-255del), which promote growth and lumen formation to a larger extent than wild-type FOXA1. Interestingly, a FOXA1 luciferase reporter assay we developed in HEK293 cells indicates that the majority of mutants tested behave as gain of function. The only mutant to score as loss of function, FOXA1R219S, is found nearly exclusively in prostate cancer, and is enriched in neuroendocrine prostate cancer cohorts. Expression of FOXA1R219S suppresses lumen formation and promotes both basal and epithelial-mesenchymal transition transcriptional programs. However, this mutant also provides a growth advantage beyond what is conferred by FOXA1WT. Taken together, these data suggest that FOXA1R219S is not simply a loss of function allele and instead may drive disease progression through a different set of signaling pathways than the FOXA1 alleles that promote luminal differentiation. Further study of these classes of mutations and how they relate to patient outcome, along with analysis of the FOXA1mut/AR cistromes, is ongoing to determine the genome-wide implications of FOXA1 alterations and how they promote prostate cancer progression. Citation Format: Elizabeth J. Adams, Elizabeth Hoover, Wouter R. Karthaus, Charles L. Sawyers. FOXA1 promotes a luminal growth program in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 976.

Role of FOXA1 in prostate cancer and therapeutic resistance

Role of FOXA1 in prostate cancer and therapeutic resistance

Abstract 1801: FOXA1, a novel regulator of neuroendocrine differentiation

Abstract Neuroendocrine prostate cancer (NEPC) is a subtype of prostate cancer that is highly aggressive and exhibits a neuroendocrine phenotype, being distinct from prostate adenocarcinoma. Cases of NEPC are predicted to increase rapidly following broader use of new-generation hormonal therapies including abiraterone and enzalutamide. Although androgen deprivation and cytokine induction have been previously suggested to induce neuroendocrine differentiation of prostate cancer, little is known regarding the underlying molecular mechanisms. FOXA1 is a forkhead box family transcription factor that has been shown to act as a pioneer factor for androgen receptor (AR), thereby defining the prostatic transcriptional program. We have recently shown that FOXA1 plays an important role in suppressing epithelial-to-mesenchymal transdifferentiation, but is often downregulated in castration-resistant prostate cancer (CRPC). Along this line, in the present study, we showed that FOXA1 loss, achieved by shRNA-mediated knockdown, led to cell morphology changes typical of neuroendocrine differentiation, accompanied by a strong increase of NEPC marker enolase2 (ENO2), while ectopic FOXA1 overexpression reverses these alterations. This was further linked to ERK phosphorylation, which has been previously reported to be up-regulated following neuroendocrine differentiation. Moreover, to understand the mechanism underlying FOXA1 regulation of neuroendocrine differentiation, we performed bioinformatics analysis and nominated interleukin 8 (IL8) as a direct target of FOXA1 and its up-regulation is critical in mediating neuroendocrine differentiation of FOXA1-depleted cells. Finally, we validated a negative correlation between FOXA1 and neuroendocrine differentiation in primary prostate cancer specimens through analysis of publically available RNA-seq data as well as direct IHC staining of various NEPC markers. In summary, we report a new role of FOXA1 as a critical inhibitor of neuroendocrine differentiation and the importance of balanced FOXA1 expressing in the maintenance of the epithelial phenotype of prostate cancer. Citation Format: Jung Kim, Hongjian Jin, Jonathan Zhao, Vamsi Parimi, Ximing Yang, Jindan Yu. FOXA1, a novel regulator of neuroendocrine differentiation. [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 1801.

Abstract 885: Epigenetic regulation of estrogen receptor transcription by the PI3K pathway in breast cancer

Abstract Mutations in the PIK3CA gene are the most frequent genomic alterations in estrogen receptor (ER)-positive breast cancers. Direct pharmacological inhibition of PI3K signaling is therefore an attractive clinical strategy and a number of PI3K pathway inhibitors are currently under clinical development. Unfortunately, although the majority of ER-positive PIK3CA-mutant patients respond, mechanisms of resistance to these inhibitors inevitably emerge. By studying both mouse models and human samples, our laboratory has previously uncovered that inhibition of PI3K pathway increases ER transcriptional activity, which in turn renders cells more susceptible to endocrine therapy. The mechanisms by which ER and PI3K signaling pathway regulate each other in breast cancer cells, however, remain elusive. To better understand the cross-talk between the PI3K pathway and the ER transcriptional program, we developed an unbiased transposon activation mutagenesis screen with the goal of identifying modulators of resistance to PI3K inhibitors in ER-positive tumors. Among the genes identified, we found a number of key regulators of ER function including the pioneer transcription factors FOXA1 and PBX1. We further confirmed that FOXA1 and PBX1 expression and transcriptional activity was enhanced upon PI3K inhibition and validated these observations in both xenograft models and samples from patients undergoing treatment with the PI3Ká inhibitor BYL719. Moreover, chromatin imunoprecipitation (ChIP)-sequencing against ER and FOXA1 demonstrated that these factors occupy the same genomic regions, and their binding is increased upon PI3K inhibition. Silencing FOXA1 or PBX1 impaired the activation of the ER-dependent transcriptional program following PI3K blockade and sensitized cells to PI3K inhibition. To better understand the role of FOXA1 and PBX1 in the ER-PI3K crosstalk, we have then studied in detail the chromatin changes upon BYL719 treatment using transposase-accessible chromatin using high-throughput sequencing (ATAC-seq) in breast cancer cells. Epigenomic profiling using ATAC-seq is also being done on patient samples collected before BYL719 administration (pre-treatment) and during therapy (on-treatment). ATAC-seq and RNA-seq data from the same patients will be integrated using novel computational approaches. These analyses will help to dissect the ER-dependent epigenetic changes occurring upon PI3K inhibition and how the cells use these chromatin modifications to adapt to the pharmacological stress. Elucidating the interconnection between the PI3K pathway and ER activity may uncover novel mechanisms of resistance to either PI3K inhibitors or endocrine therapy in ER-positive breast cancer patients. Citation Format: Eneda Toska, Hatice Osmanbeyoglu, Moshe Elkabets, Carmen Chan, Pau Castel, Maura Dickler, Scott Armstrong, Christina Leslie, Maurizio Scaltriti, Baselga José. Epigenetic regulation of estrogen receptor transcription by the PI3K pathway in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 885.

Looking beyond Androgen Receptor Signaling in the Treatment of Advanced Prostate Cancer

This review will provide a description of recent efforts in our laboratory contributing to a general goal of identifying critical determinants of prostate cancer growth in both androgen-dependent and -independent contexts. Important outcomes to date have indicated that the sustained activation of AR transcriptional activity in castration-resistant prostate cancer (CRPC) cells results in a gene expression profile separate from the androgen-responsive profile of androgen-dependent prostate cancer (ADPC) cells. Contributing to this reprogramming is enhanced FoxA1 recruitment of AR to G2/M phase target gene loci and the enhanced chromatin looping of CRPC-specific gene regulatory elements facilitated by PI3K/Akt-phosphorylated MED1. We have also observed a role for FoxA1 beyond AR signaling in driving G1/S phase cell cycle progression that relies on interactions with novel collaborators MYBL2 and CREB1. Finally, we describe an in-depth mechanism of GATA2-mediated androgen-responsive gene expression in both ADPC and CRPC cells. Altogether these efforts provide evidence to support the development of novel prostate cancer therapeutics that address downstream targets of AR activity as well as AR-independent drivers of disease-relevant transcription programs.

Fine-Scale Mapping of the FGFR2 Breast Cancer Risk Locus: Putative Functional Variants Differentially Bind FOXA1 and E2F1

The 10q26 locus in the second intron of FGFR2 is the locus most strongly associated with estrogen-receptor-positive breast cancer in genome-wide association studies. We conducted fine-scale mapping in case-control studies genotyped with a custom chip (iCOGS), comprising 41 studies (n = 89,050) of European ancestry, 9 Asian ancestry studies (n = 13,983), and 2 African ancestry studies (n = 2,028) from the Breast Cancer Association Consortium. We identified three statistically independent risk signals within the locus. Within risk signals 1 and 3, genetic analysis identified five and two variants, respectively, highly correlated with the most strongly associated SNPs. By using a combination of genetic fine mapping, data on DNase hypersensitivity, and electrophoretic mobility shift assays to study protein-DNA binding, we identified rs35054928, rs2981578, and rs45631563 as putative functional SNPs. Chromatin immunoprecipitation showed that FOXA1 preferentially bound to the risk-associated allele (C) of rs2981578 and was able to recruit ERα to this site in an allele-specific manner, whereas E2F1 preferentially bound the risk variant of rs35054928. The risk alleles were preferentially found in open chromatin and bound by Ser5 phosphorylated RNA polymerase II, suggesting that the risk alleles are associated with changes in transcription. Chromatin conformation capture demonstrated that the risk region was able to interact with the promoter of FGFR2, the likely target gene of this risk region. A role for FOXA1 in mediating breast cancer susceptibility at this locus is consistent with the finding that the FGFR2 risk locus primarily predisposes to estrogen-receptor-positive disease.

Androgen Receptor-Independent Function of FoxA1 in Prostate Cancer Metastasis

FoxA1 (FOXA1) is a pioneering transcription factor of the androgen receptor (AR) that is indispensible for the lineage-specific gene expression of the prostate. To date, there have been conflicting reports on the role of FoxA1 in prostate cancer progression and prognosis. With recent discoveries of recurrent FoxA1 mutations in human prostate tumors, comprehensive understanding of FoxA1 function has become very important. Here, through genomic analysis, we reveal that FoxA1 regulates two distinct oncogenic processes via disparate mechanisms. FoxA1 induces cell growth requiring the AR pathway. On the other hand, FoxA1 inhibits cell motility and epithelial-to-mesenchymal transition (EMT) through AR-independent mechanism directly opposing the action of AR signaling. Using orthotopic mouse models, we further show that FoxA1 inhibits prostate tumor metastasis in vivo. Concordant with these contradictory effects on tumor progression, FoxA1 expression is slightly upregulated in localized prostate cancer wherein cell proliferation is the main feature, but is remarkably downregulated when the disease progresses to metastatic stage for which cell motility and EMT are essential. Importantly, recently identified FoxA1 mutants have drastically attenuated ability in suppressing cell motility. Taken together, our findings illustrate an AR-independent function of FoxA1 as a metastasis inhibitor and provide a mechanism by which recurrent FoxA1 mutations contribute to prostate cancer progression.

Insight into mechanism of in vitro insulin secretion increase induced by antipsychotic clozapine: Role of FOXA1 and mitochondrial citrate carrier

The use of clozapine and other antipsychotic drugs is known to be associated with a number of adverse metabolic side effects, including diabetes mellitus. These side effects could be, at least in part, the result of impaired islet cell function and abnormal insulin secretion, although the underlying mechanisms are unknown. The aim of this study is the identification of targets for clozapine related to the abnormal insulin secretion. We identify a specific activation of the transcriptional factor FOXA1, but not FOXA2 and FOXA3, by clozapine in HepG2 cells. Clozapine enhances FOXA1 DNA-binding and its transcriptional activity, increasing mitochondrial citrate carrier gene expression, which contains a FOXA1 site in its promoter. Haloperidol, a conventional antipsychotic drug, does not determine any increase of FOXA1 gene expression. We also demonstrate that clozapine upregulates FOXA1 and CIC gene expression in INS-1 cells only at basal glucose concentration.In addition, we find that abnormal insulin secretion in basal glucose conditions could be completely abolished by FOXA1 silencing in INS-1 cells treated with clozapine.The identification of FOXA1 as a novel target for clozapine may shed more light to understand molecular mechanism of abnormal insulin secretion during clozapine treatment.

A Role for Forkhead Box A1 in Acute Lung Injury

Forkhead box protein A1 (FoxA1) is an evolutionarily conserved winged helix transcription factor with diverse regulatory functions. However, little is known about the role of FoxA1 in acute lung injury (ALI) and pulmonary cell injury. In this study, an in vivo model was employed whereby rats were administered an intravenous injection of oleic acid (OA, 0.1 ml/kg), and alveolar type II epithelial cells (AT-2 cells) injury was induced by hydrogen peroxide (H(2)O(2)) in vitro. OA injection resulted in lung injury and AT-2 cells apoptosis in vivo. OA injection and H(2)O(2) upregulated FoxA1 mRNA and protein in lung tissue of the in vivo ALI model and in H(2)O(2) challenged AT-2 cells. Overexpression of FoxA1 promoted apoptosis, whereas FoxA1 deficiency, induced by antisense oligonucleotides, decreased AT-2 cells apoptosis induced by H(2)O(2), as shown by flow cytometry. These results suggest that FoxA1 may play an important role in ALI by promoting apoptosis of pulmonary epithelial cells.

Up-regulation of the HSP72 by Foxa1 in MCF-7 human breast cancer cell line

Forkhead box protein A1 (Foxa1) is an evolutionarily conserved winged helix transcription factor. In this study, the effect of Foxa1 on the expression of HSP72 was examined by RT-PCR and Western blot in Foxa1 overexpression or deficient cells. The results showed overexpression of Foxa1 promoted the expression of HSP72, while Foxa1 depletion, induced by antisense oligonucleotides, decreased the expression of HSP72 in MCF-7 cells under normal and heat stress condition. Electrophoretic mobility shift assay and chromatin immunoprecipitation revealed that Foxa1 bound to HSP72 promoter, and heat stress promoted its DNA binding activity. Luciferase reporter showed that Foxa1 also increased the transcription activity of HSP72 promoter. These results indicate an important role for Foxa1 as a novel regulator of expression of HSP72.

Role of Foxa1 in regulation of bcl2 expression during oxidative-stress-induced apoptosis in A549 type II pneumocytes

Forkhead box protein A1 (Foxa1) is an evolutionarily conserved winged helix transcription factor that was traditionally considered to be involved in embryonic development and cell differentiation. However, little is known about the role of Foxa1 in oxidative-stress-induced apoptosis. In this study, hydrogen peroxide (H(2)O(2))-induced apoptosis, upregulation of Foxa1, and the role of Foxa1 in the regulation of bcl2 gene expression were studied in A549 type II pneumocytes. H(2)O(2) upregulated Foxa1 mRNA and protein in a time- and dose-dependent manner. Overexpression of Foxa1 promoted apoptosis, whereas Foxa1 deficiency, induced by antisense oligonucleotides, decreased A549 cell apoptosis induced by H(2)O(2), as shown by flow cytometry. Moreover, Foxa1 overexpression decreased the expression of bcl2, while Foxa1 depletion increased the expression of bcl2. Electrophoretic mobility shift assay and chromatin immunoprecipitation revealed that Foxa1 bound to bcl2 promoter, and H(2)O(2) promoted its DNA binding activity. Luciferase reporter showed that Foxa1 also decreased the transcription activity of bcl2 promoter under normal conditions and oxidative stress. These results indicate that Foxa1 plays a pro-apoptotic role by inhibiting the expression of anti-apoptotic gene bcl2.