Cristian P. Moiola

Transcriptional Autoregulation by BRCA1

The BRCA1 gene product plays numerous roles in regulating genome integrity. Its role in assembling supermolecular complexes in response to DNA damage has been extensively studied; however, much less is understood about its role as a transcriptional coregulator. Loss or mutation is associated with hereditary breast and ovarian cancers, whereas altered expression occurs frequently in sporadic forms of breast cancer, suggesting that the control of BRCA1 transcription might be important to tumorigenesis. Here, we provide evidence of a striking linkage between the roles for BRCA1 as a transcriptional coregulator with control of its expression via an autoregulatory transcriptional loop. BRCA1 assembles with complexes containing E2F-1 and RB to form a repressive multicomponent transcriptional complex that inhibits BRCA1 promoter transcription. This complex is disrupted by genotoxic stress, resulting in the displacement of BRCA1 protein from the BRCA1 promoter and subsequent upregulation of BRCA1 transcription. Cells depleted of BRCA1 respond by upregulating BRCA1 transcripts, whereas cells overexpressing BRCA1 respond by downregulating BRCA1 transcripts. Tandem chromatin immmunoprecipitation studies show that BRCA1 is regulated by a dynamic coregulatory complex containing BRCA1, E2F1, and Rb at the BRCA1 promoter that is disrupted by DNA-damaging agents to increase its transcription. These results define a novel transcriptional mechanism of autoregulated homeostasis of BRCA1 that selectively titrates its levels to maintain genome integrity in response to genotoxic insult.

Identification of new Rel/NF-kappaB regulatory networks by focused genome location analysis

NF-κB is an inducible transcription factor that controls kinetically complex patterns of gene expression. Several studies reveal multiple pathways linking NF-κB to the promotion and progression of various cancers. Despite extensive interest and characterization, many NF-κB controlled genes still remain to be identified. We used chromatin immunoprecipitation combined with microarray technology (ChIP/Chip) to investigate the dynamic interaction of NF-κB with the promoter regions of 100 genes known to be expressed in mitogen-induced T-cells. Six previously unrecognized NF-κB controlled genes (ATM, EP300, TGFβ, Selectin, MMP-1, and SFN) were identified. Each gene is induced in mitogen-stimulated T-cells, repressed by pharmacological NF-κB blockade, reduced in cells deficient in the p50 NF-κB subunit and dramatically repressed by RNAi specifically designed against cRel. A coregulatory role for Ets transcription factors in the expression of the NF-κB controlled genes was predicted by comparative promoter analysis and confirmed by ChIP and by functional disruption of Ets. NF-κB deficiency produces a deficit in ATM function and DNA repair indicating an active role for NF-κB in maintaining DNA integrity. These results define new potential targets and transcriptional networks governed by NF-κB and provide novel functional insights for the role of NF-κB in genomic stability, cell cycle control, cell-matrix and cell-cell interactions during tumor progression.

BRCA1 loss induces GADD153-mediated doxorubicin resistance in prostate cancer.

BRCA1 plays numerous roles in the regulation of genome integrity and chemoresistance. Although BRCA1 interaction with key proteins involved in DNA repair is well known, its role as a coregulator in the transcriptional response to DNA damage remains poorly understood. In this study, we show that BRCA1 plays a central role in the transcriptional response to genotoxic stress in prostate cancer. BRCA1 expression mediates apoptosis, cell-cycle arrest, and decreased viability in response to doxorubicin treatment. Xenograft studies using human prostate carcinoma PC3 cells show that BRCA1 depletion results in increased tumor growth. A focused survey of BRCA1-regulated genes in prostate carcinoma reveals that multiple regulators of genome stability and cell-cycle control, including BLM, FEN1, DDB2, H3F3B, BRCA2, CCNB2, MAD2L1, and GADD153, are direct transcriptional targets of BRCA1. Furthermore, we show that BRCA1 targets GADD153 promoter to increase its transcription in response to DNA damage. Finally, GADD153 depletion significantly abrogates BRCA1 influence on cell-cycle progression and cell death in response to doxorubicin treatment. These findings define a novel transcriptional pathway through which BRCA1 orchestrates cell fate decisions in response to genotoxic insults, and suggest that BRCA1 status should be considered for new chemotherapeutic treatment strategies in prostate cancer. Mol Cancer Res; 9(8); 1078–90. ©2011 AACR.

Cyclin T1 overexpression induces malignant transformation and tumor growth

Human PTEFb is a protein kinase composed by CDK9 and Cyclin T that controls the elongation phase of RNA Pol II. This complex also affects the activation and differentiation program of lymphoid cells. In this study we found that several head and neck tumor cell lines overexpress PTEFb. We also established that Cyclin T1 is able to induce transformation in vitro, as we determined by foci and colony formation assays. Nu/nu mice s.c. injected with stable transfected Cyclin T1 cells (NIH 3T3 Cyclin T1) developed tumors faster than animals injected with control cells (NIH 3T3 b-gal). In vitro, NIH 3T3 Cyclin T1 cells show increased proliferation and CDK4-Rb phosphorylation. Even more, silencing E2F1 expression (shRNA E2F1) in NIH 3T3 cells resulted in a dramatic inhibition of Cyclin T1-induced foci. All these data demonstrate for the first time the Cyclin T1 oncogenic function and suggest a role for this protein in controlling cell cycle probably via Rb/E2F1 pathway.

Dynamic Coregulatory Complex Containing BRCA1, E2F1 and CtIP Controls ATM Transcription

Chromosomal instability is a key feature in cancer progression. Recently we have reported that BRCA1 regulates the transcription of several genes in prostate cancer, including ATM (ataxia telangiectasia mutated). Although it is well accepted that ATM is a pivotal mediator in genotoxic stress, it is unknown whether ATM transcription is regulated during the molecular response to DNA damage. Here we investigate ATM transcription regulation in human prostate tumor PC3 cell line. We have found that doxorubicin and mitoxantrone repress ATM transcription in PC3 cells but etoposide and methotrexate do not affect ATM expression. We have demonstrated that BRCA1 binds to ATM promoter and after doxorubicin exposure, it is released. BRCA1 overexpression increases ATM transcription and this enhancement is abolished by BRCA1 depletion. Moreover, BRCA1-BRCT domain loss impairs the ability of BRCA1 to regulate ATM promoter activity, strongly suggesting that BRCT domain is essential for ATM regulation by BRCA1. BRCA1-overexpressing PC3 cells exposed to KU55933 ATM kinase inhibitor showed significant decreased ATM promoter activity compared to untreated cells, suggesting that ATM transcriptional regulation by BRCA1 is partially mediated by the ATM kinase activity. In addition, we have demonstrated E2F1 binding to ATM promoter before and after doxorubicin exposure. E2F1 overexpression diminishes ATM transcription after doxorubicin exposure which is impaired by E2F1 dominant negative mutants. Finally, the co-regulator of transcription CtIP increases ATM transcription. CtIP increases ATM transcription. Altogether, BRCA1/E2F1/CtIP binding to ATM promoter activates ATM transcription. Doxorubicin exposure releases BRCA1 and CtIP from ATM promoter still keeping E2F1 recruited and, in turn, represses ATM expression.

CtBP1 associates metabolic syndrome and breast carcinogenesis targeting multiple miRNAs

Metabolic syndrome (MeS) has been identified as a risk factor for breast cancer. C-terminal binding protein 1 (CtBP1) is a co-repressor of tumor suppressor genes that is activated by low NAD+/NADH ratio. High fat diet (HFD) increases intracellular NADH. We investigated the effect of CtBP1 hyperactivation by HFD intake on mouse breast carcinogenesis. We generated a MeS-like disease in female mice by chronically feeding animals with HFD. MeS increased postnatal mammary gland development and generated prominent duct patterns with markedly increased CtBP1 and Cyclin D1 expression. CtBP1 induced breast cancer cells proliferation. Serum from animals with MeS enriched the stem-like/progenitor cell population from breast cancer cells. CtBP1 increased breast tumor growth in MeS mice modulating multiple genes and miRNA expression implicated in cell proliferation, progenitor cells phenotype, epithelial to mesenchymal transition, mammary development and cell communication in the xenografts. These results define a novel function for CtBP1 in breast carcinogenesis.

Association of HO-1 and BRCA1 Is Critical for the Maintenance of Cellular Homeostasis in Prostate Cancer

Prostate cancer is the second leading cause of cancer-related death in men worldwide. Many factors that participate in the development of prostate cancer promote imbalance in the redox state of the cell. Accumulation of reactive oxygen species causes injury to cell structures, ultimately leading to cancer development. The antioxidant enzyme heme oxygenase 1 (HMOX1/HO-1) is responsible for the maintenance of the cellular homeostasis, playing a critical role in the oxidative stress and the regulation of prostate cancer development and progression. In the present study, the transcriptional regulation of HO-1 was investigated in prostate cancer. Interestingly, the tumor suppressor BRCA1 binds to the HO-1 promoter and modulates HO-1, inducing its protein levels through both the increment of its promoter activity and the induction of its transcriptional activation. In addition, in vitro and in vivo analyses show that BRCA1 also controls HO-1–negative targets: MMP9, uPA, and Cyclin D1. HO-1 transcriptional regulation is also modulated by oxidative and genotoxic agents. Induction of DNA damage by mitoxantrone and etoposide repressed HO-1 transcription, whereas hydrogen peroxide and doxorubicin induced its expression. Xenograft studies showed that HO-1 regulation by doxorubicin also occurs in vivo. Immunofluorescence analysis revealed that BRCA1 overexpression and/or doxorubicin exposure induced the cytoplasmic retention of HO-1. Finally, the transcription factor NRF2 cooperates with BRCA1 protein to activate HO-1 promoter activity. In summary, these results show that the activation of BRCA1–NRF2/HO-1 axis defines a new mechanism for the maintenance of the cellular homeostasis in prostate cancer. Implications: Oxidative and genotoxic stress converge on HO-1 transcriptional activity through the combined actions of BRCA1 and NRF2. Mol Cancer Res; 13(11); 1455–64. ©2015 AACR.

ALCAM shedding at the invasive front of the tumor is a marker of myometrial infiltration and promotes invasion in endometrioid endometrial cancer

Endometrial cancer (EC) is the sixth deadliest cancer in women. The depth of myometrial invasion is one of the most important prognostic factors, being directly associated with tumor recurrence and mortality. In this study, ALCAM, a previously described marker of EC recurrence, was studied by immunohistochemistry at the superficial and the invasive tumor areas from 116 EC patients with different degree of myometrial invasion and related to a set of relevant epithelial and mesenchymal markers. ALCAM expression presented a heterogeneous functionality depending on its localization, it correlated with epithelial markers (E-cadherin/β-catenin) at the superficial area, and with mesenchymal markers at the invasive front (COX-2, SNAIL, ETV5, and MMP-9). At the invasive front, ALCAM-negativity was an independent marker of myometrial invasion. This negativity, together with an increase of soluble ALCAM in uterine aspirates from patients with an invasive EC, and its positive correlation with MMP-9 levels, suggested that ALCAM shedding by MMP-9 occurs at the invasive front. In vivo and in vitro models of invasive EC were generated by ETV5-overexpression. In those, we demonstrated that ALCAM shedding was related to a more invasive pattern and that full-ALCAM recovery reverted most of the ETV5-cells mesenchymal abilities, partially through a p-ERK dependent-manner.

Abstract 1160: CtBP1 is the molecular link that associates breast cancer and metabolic syndrome

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Breast cancer is still one of the most important public health problems in the entire world. Obesity and metabolic syndrome (MS) increases the incidence and aggressiveness of breast cancer. C-Terminal Binding Protein (CtBP1) is a transcriptional corepressor of tumor suppressor genes and is considered a molecular sensor of cell metabolic state due to is activated in high energy conditions (high NADH). In this work we studied the effects of the activation of CtBP1 pathway by metabolic syndrome on breast tumor development and progression. We generated a murine model of MS by chronic high fat diet (HFD) administration. By histological and whole mount methods, we found that breast tissue of animals receiving HFD presented higher levels of immature adipose tissue and an increased glandular area with more generation of lateral branches and terminal end buds of mammary ducts. Breast tissue of HFD animals also showed higher expression of the proliferation markers (cyclin D1) and epithelial markers (E-cadherin). Interestingly, HFD induced CtBP1 expression in the mammary ducts. Furthermore, the number and size of mamospheres generated with LM38-LP breast cancer cells were significantly increased when cells were incubated with serum from HFD fed mice compared to the serum of animals under control diet (CD). In addition, to investigate CtBP1 role in tumor progression we performed xenografts in nude mice fed with CD or HFD by subcutaneous injecton of breast tumor cells MDA MB 231 with depleted CtBP1 expression (shRNA CtBP1) or control cells (shRNA scramble). We found that CtBP1 depletion dramatically decreased tumor growth and KI67 expression relative to control tumors. Furthermore, xenografts developed in HFD fed mice were less differentiated compared to CD. Finally, CtBP1 diminished expression tumors showed lower mesenchymal markers expression, progenitor cells markers and markers involved in mammary development. Our studies demonstrated for the first time that gene transcription regulation by CtBP1 provides an important molecular link among MS, CtBP1 function and tumor growth. Hence, these results suggest an association to understand metabolism and breast cancer. Citation Format: Paola De Luca, Nicolas Dalton, Cristian Pablo Moiola, Carolina Flumian, Georgina Scalise, Juliana Porretti, Cintia Massillo, Edith Kordon, Laura Todaro, Elba Vazquez, Roberto Meiss, Adriana De Siervi. CtBP1 is the molecular link that associates breast cancer and metabolic syndrome. [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 1160. doi:10.1158/1538-7445.AM2015-1160

Abstract 3697: Molecular link that associates high fat diet and prostate tumor growth.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Prostate Cancer (PCa) is one of the most common invasive tumors in men. Epidemiological studies indicate that diet and overweight are important factors implicated in prostate carcinogenesis. Obesity is associated with PCa aggressiveness, poorer prognosis and increased mortality. Breast cancer susceptibility gene 1 (BRCA1) interacts with several transcriptional regulators to modulate the androgen receptor (AR) signaling in PCa cell lines. Germline mutations in this gene increase breast cancer risk and are associated with high grade PCa. Previously, it had been reported that C-terminal Binding Protein 1 (CtBP1) acts as a switch to control BRCA1 transcription in response to the metabolic status of the cells. The release of CtBP1 from BRCA1 promoter through estrogen induction and high NAD+/NADH ratio (similar to high caloric intake) increases BRCA1 transcription in breast cancer cells. The aim of this work was to assess the effect of androgens and/or high fat diet over the BRCA1/CtBP1 axis and PCa tumor growth. We found that BRCA1 and CtBP1 proteins associate to BRCA1 proximal promoter region in PC3 cells and suppress BRCA1 transcription. Testosterone stimulation released these factors from BRCA1 promoter increasing its transcription. To assess whether this activation is mediated by testosterone or the estrogen, synthesized from testosterone by the aromatase (CYP19A1), we investigated this mechanism in the presence of letrozol (LTZ), an aromatase inhibitor. We found that LTZ abolished BRCA1 induction by testosterone, suggesting that BRCA1 activation is mediated by estrogen in these cells. Furthermore, we generated PC3 cell lines transfected with pcDNA3-CtBP1 (PC3-CtBP1) or shRNA-CtBP1 (PC3-shCtBP1) plasmids, to overexpress or knock down CtBP1 expression, respectively. CtBP1 induction decreased BRCA1 expression in these cells and this effect was reverted by CtBP1 depletion. In addition, PC3-CtBP1 cells showed increased clonogenic capacity and proliferation compared to PC3-shCtBP1 cells. Moreover, we developed an in vivo model to investigate the effect of high caloric diet on PCa growth after CtBP1 modulated-expression. High fat or control diet fed male nude mice were inoculated with PC3-CtBP1 and PC3-shCtBP1 stable cells. We found that CtBP1 depleted cells growing as xenografts in high fat diet fed mice dramatically decreased prostate tumor growth. Molecular analysis of tumors by RT-qPCR showed that CtBP1 depletion correlated with high BRCA1 expression. In addition, serum from high fat fed mice significantly induced PC3-CtBP1 cell proliferation in vitro. These results strongly suggest that the potential oncogenic role of CtBP1 is dependent on the caloric diet intake. Hence, BRCA1 regulation by CtBP1 provides an important molecular link between caloric intake and tumor suppressor expression. Citation Format: Cristian P. Moiola, Paola De Luca, Florencia Zalazar, Javier Cotignola, Estefania Labanca, Roberto Meiss, Elba S. Vazquez, Kevin Gardner, Adriana De Siervi. Molecular link that associates high fat diet and prostate tumor growth. [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 3697. doi:10.1158/1538-7445.AM2013-3697