Jay Vadgama

Down-Regulation and Growth Inhibitory Role of C/EBPα in Breast Cancer

Purpose: CCAAT/enhancer binding proteins (C/EBP) are a family of transcription factors that regulate proliferation and differentiation in a variety of tissues. The purpose of this study was to explore the possibility that C/EBPα is involved in breast cancer. Experimental Design: We quantified C/EBPα mRNA expression levels in 24 primary breast tumors, 16 normal breast samples, and 8 breast cancer cell lines using quantitative real-time reverse transcription-PCR assay. C/EBPα protein levels were further determined by immunohistochemical analysis. To examine the consequence of C/EPBα expression in breast cancer, we stably transfected an inducible C/EPBα expression vector into three breast cancer cell lines. Results: Low expression of C/EBPα mRNA was found in 83% of primary breast cancer samples. Immunohistochemical study further showed either a markedly reduced or undetectable expression of C/EBPα protein in 30% of breast cancer specimens. The other 70% of breast cancers had C/EBPα expression in both the cytoplasm and nucleus; in control, C/EBPα was localized to the nucleus in the normal ductal cells. C/EBPα expression was associated with estrogen- and progesterone receptor–negative status. Induction of C/EBPα expression in these cell lines resulted in growth inhibition accompanied by G0-G1 cell cycle arrest and reduced anchorage-independent cell growth. C/EBPα expression was associated with down-regulation of c-myc and up-regulation of p21, PPARγ, and the breast epithelial differentiation marker, maspin. Conclusions: These results suggest that reduced expression of C/EBPα may play a role in the development and/or progression of breast cancer.

Expression of connective tissue growth factor (CTGF/CCN2) in breast cancer cells is associated with increased migration and angiogenesis.

Connective tissue growth factor (CTGF/CCN2) belongs to the CCN family of matricellular proteins, comprising Cyr61, CTGF, NovH and WISP1-3. The CCN proteins contain an N-terminal signal peptide followed by four conserved domains sharing sequence similarities with the insulin-like growth factor binding proteins, von Willebrand factor type C repeat, thrombospondin type 1 repeat, and a C-terminal growth factor cysteine knot domain. To investigate the role of CCN2 in breast cancer, we transfected MCF-7 cells with full-length CCN2, and with four mutant constructs in which one of the domains had been deleted. MCF-7 cells stably expressing full-length CCN2 demonstrated reduced cell proliferation, increased migration in Boyden chamber assays and promoted angiogenesis in chorioallantoic membrane assays compared to control cells. Deletion of the C-terminal cysteine knot domain, but not of any other domain-deleted mutants, abolished activities mediated by full-length CCN2. We have dissected the role of CCN2 in breast tumorigenesis on a structural basis.

Connective tissue growth factor associated with oncogenic activities and drug resistance in glioblastoma multiforme

Connective tissue growth factor (CTGF or CCN2) is a secreted protein that belongs to the CCN [cysteine‐rich CYR61/CTGF/nephroblastoma‐overexpressed gene] family. These proteins have been implicated in various biological processes, including stimulation of cell proliferation, migration, angiogenesis and tumorigenesis. In a previous study, we found that CTGF mRNA was elevated in primary gliomas, and a significant correlation existed between CTGF mRNA levels versus tumor grade, histology and patient survival. In this study, the role of CTGF in glioma tumorigenesis was explored. Forced expression of CTGF in glioblastoma multiforme (GBM) cells accelerated their growth in liquid culture and soft agar, stimulated cells migration in Boyden chamber assays and significantly increased their ability to form large, vascularized tumors in nude mice. CTGF induced the expression of the antiapoptotic proteins, Bcl‐xl, Survivin and Flip. Overexpression of CTGF caused the U343 GBM cells to survive for longer than 40 days in serum‐free medium and resist antitumor drugs including tumor necrosis factor (TNF), TNF‐related apoptosis‐inducing ligand, VELCADE (bortezomib, proteasome inhibitor) and temozolomide. Our data suggest that CTGF plays an important role in glioma progression, by supporting tumor cells survival and drug resistance.

Novel Gemini-vitamin D3 analog inhibits tumor cell growth and modulates the Akt/mTOR signaling pathway.

We have shown previously that 1alpha, 25-dihydroxy-21-(3-hydroxy-3-methylbutyl)vitamin D3 (Gemini) compounds, which have two side chains attached to carbon-20, had increased anti-tumor activities against breast, prostate and leukemia cell lines in comparison to 1,25(OH)2 vitamin D3. This prompted us to synthesize additional Gemini compounds with further modifications and evaluate their anticancer effects. Most effective in this series was 1,25-dihydroxy-20S-21(3-hydroxy-3-methyl-butyl)-23-yne-26,27-hexafluoro-vitamin D3 [Gemini-23-yne-26,27-hexafluoro-D3]. This analog was approximately 10-fold more potent than previously characterized Gemini compounds in inhibiting the clonal growth of HL-60, MCF-7 and LNCaP cell lines. Also in MCF-7 cells, Gemini-23-yne-26,27-hexafluoro-D3 caused dephosphorylation of the oncogenic kinase, Akt, resulting in dephosphorylation of the Akt target proteins, Forkhead transcription factor and mammalian target of rapamycin (mTOR). Downstream effectors of mTOR were also inhibited by the analog as demonstrated by decreased phosphorylation of both S6 kinase, and the translation inhibitor, 4E-BP1. The mTOR pathway regulates mRNA translation; exposure of MCF-7 cells to Gemini-23-yne-26,27-hexafluoro-D3 decreased their rate of protein synthesis and increased the association of 4EBP-1 with the translation initiation factor, eIF4E. Inhibition of the Akt-mTOR pathway represents a novel mechanism by which vitamin D3 analogs may modulate the expression and activity of proteins involved in cancer cell proliferation.

Genomic and functional characterizations of phosphodiesterase subtype 4D in human cancers

Discovery of cancer genes through interrogation of genomic dosage is one of the major approaches in cancer research. In this study, we report that phosphodiesterase subtype 4D (PDE4D) gene was homozygously deleted in 198 cases of 5,569 primary solid tumors (3.56%), with most being internal microdeletions. Unexpectedly, the microdeletions did not result in loss of their gene products. Screening PDE4D expression in 11 different types of primary tumor samples (n = 165) with immunohistochemistry staining revealed that its protein levels were up-regulated compared with corresponding nontransformed tissues. Importantly, depletion of endogenous PDE4D with three independent shRNAs caused apoptosis and growth inhibition in multiple types of cancer cells, including breast, lung, ovary, endometrium, gastric, and melanoma, which could be rescued by reexpression of PDE4D. We further showed that antitumor events triggered by PDE4D suppression were lineage-dependently associated with Bcl-2 interacting mediator of cell death (BIM) induction and microphthalmia-associated transcription factor (MITF) down-regulation. Furthermore, ectopic expression of the PDE4D short isoform, PDE4D2, enhanced the proliferation of cancer cells both in vitro and in vivo. Moreover, treatment of cancer cells with a unique specific PDE4D inhibitor, 26B, triggered massive cell death and growth retardation. Notably, these antineoplastic effects induced by either shRNAs or small molecule occurred preferentially in cancer cells but not in nonmalignant epithelial cells. These results suggest that although targeted by genomic homozygous microdeletions, PDE4D functions as a tumor-promoting factor and represents a unique targetable enzyme of cancer cells.

Abstract 5559: Long-term exposure of breast cell lines to ethanol affects the transcriptional signature for some oncogenic gene families, but has little effect on this phenotype in mammospheres or on the expression of stem cell markers

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Objectives. Chronic alcoholism is a risk factor for breast cancer but the mechanism of its potential oncogenic effects is unknown. Stem cells have been found in the epithelium of normal breast tissue and are postulated to convert into cancer stem cells driving tumor generation and metastasis. Breast stem cells are assumed to be enriched within multiple-cell spheroids named mammospheres. Here we aimed to determine whether ethanol, or its metabolite, acetaldehyde, stimulate the in vitro proliferation and malignant transformation of human breast stem cells within epithelial cell cultures and mammospheres, and whether this process is defined by specific gene expression signatures denoting oncogenesis, stemness and/or alcohol-regulation. Methods. Cultures of monolayer attached cells and mammospheres from MCF-12 (“normal”) and MCF-7 (“malignant”) cell lines were incubated +/- 25 mM ethanol and subjected to DNA microarray analysis, or in triplicate with increasing concentrations of ethanol (0-25 mM) or acetaldehyde (0-12.5 mM) for 6 days, followed by immunocytofluorescence, quantitative western blot for stem cell markers Oct4 and nanog, and by RT-PCR, the formazan assay and growth in soft agar. Results. Low levels of mRNAs for stem cell markers were seen by DNA microarrays in all cultures, but except for Sox4 and Jag1 were not responsive to ethanol. Oct4 and nanog proteins were detected in the nuclei of MCF-7 attached cells, and expressed respectively as 45 (Oct4a) and 38 kDa bands for the active isoforms, whereas in MCF-12 attached cells they were detected as the variant 33 (Oct4b) and 55 kDa proteins. Ethanol reduced Oct4a moderately in MCF-7 attached cells and acetaldehyde increased it, but no changes occurred in mammospheres. Ethanol increased Oct4b and reduced nanog in MCF-12 attached cells, with no change (Oct4) or an increase (nanog) in mammospheres. In both MCF-7 and MCF-12 attached cells, but not in mammospheres, ethanol upregulated by >2 several alcohol-responsive metallothioneins (mainly 1F, 1X, and 2A), and related trans-membrane proteases serine (TMPRSS), as well as centromer-associated protein and PAI 1, while reducing other serpines and caspase 4, all malignancy-associated genes. Conclusions. Although stem cell markers were expressed in MCF-7 and MCF-12 attached cells, they were not increased in mammospheres, raising doubts about the putative stem cell enrichment in these spheroids. The inconsistent modulation of stem cell number by ethanol and acetaldehyde requires confirmation by cell sorting. The identification of several ethanol-responsive, malignancy-associated genes in both MCF-7 and MCF-12 attached cells may support an oncogenic risk for high alcohol levels. 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 5559. doi:10.1158/1538-7445.AM2011-5559

Abstract 704: Expression of FOXO1 is associated with GATA3 and Annexin-1 and predicts disease-free survival in breast cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Purpose: To determine the prognostic value of FOXO1, GATA3 and Annexin-1 expression in breast cancer. Methods: Tissue microarray and individual paraffin tissue slides from 131 patients were used for the study. The association of FOXO1, GATA3 and Annexin-1 expression with clinicopathological features of breast cancer and disease outcome was examined in retrospective samples. Kaplan-Meier survival curves and Cox regression with multivariate analysis were used for assessing the relative risk (RR) and disease-free survival (DFS). The expression of FOXO1, GATA3 and Annexin-1 were determined by immunohistochemistry and the association among the three proteins was analyzed by Logistic regression analysis. Results: The nuclear expression of FOXO1 was observed in most of the normal breast tissues and 51.3% of the malignant breast tissues. GATA3 and Annexin-1 were expressed at 73% and 24.6% respectively in breast cancer tissues. The expression of FOXO1, GATA3 and Annexin-1 were all inversely correlated with lymph node-positive tumors. Both FOXO1 and Annexin-1 expression were also inversely associated with HER2-overexpressing tumors. FOXO1 expression was significantly associated with both GATA3 and Annexin-1 expression. In addition, Multivariate analyses confirm that only FOXO1 levels independently predict DFS. Conclusion: FOXO1 expression in breast cancer is regulated by the PI3K/Akt pathway. The expression of FOXO1 is also associated with GATA3 and/or Annexin-1. Restoring or targeting FOXO1 to the cell nucleus in breast cancer tissues may improve response to therapy and disease outcome. Further clinical studies are warranted to test this hypothesis. 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 704. doi:1538-7445.AM2012-704

Abstract B68: EGFR signaling mediated modulation of transcription and probable crosstalk with components of Wnt signaling

Introduction: The purpose of the study is to examine the crosstalk between HER2/EGFR and Wnt signaling in HER2-positive (HER2+) breast cancer cells. Human epidermal growth factor receptors (HER) constitute a family of four transmembrane receptors (HER1-HER4). Ligand binding to HER1 (EGFR), HER3, and HER4 results in heterodimerization with other HER family members, including HER2. HER2+ breast cancer is characterized by an amplification of the HER2 gene, resulting in an increase in HER2 protein presence on the surface of cells, magnification of downstream intracellular signaling, and enhanced responsiveness to ligand stimulation (e.g., EGF). Approximately 20-30% of breast cancers have HER2 amplifications. The Wnt pathway is highly conserved in mammals and overexpression of some Wnt family members results in cancer. Wnts are ligands that bind to Frizzled/LRP receptors to initiate downstream signaling that results in the stabilization and nuclear translocation of β-catenin. Once in the nucleus, β-catenin associates with activators such as TCF/LEF, SMADs, ATF2, and KLF4 to promote the transcription of many target genes. EGFR and Wnt crosstalk has been observed in various cell types following EGF treatment. As an example, EGF treatment of human epidermoid carcinoma cells results in β-catenin nuclear translocation and activation of TCF/LEF dependent reporters. Mitogen-Activated Protein Kinases (MAPKs), which are activated following EGF stimulation, have been demonstrated to inhibit GSK3β, a negative regulator of β-catenin. However, no genome-wide analysis has been conducted to determine what genes are regulated by β-catenin following EGF treatment in HER2+ breast cancer cells. We sought to determine the modulation of gene expression following the stimulation of HER2+ breast cancer cells with EGF and to investigate the mechanisms that underlie the changes observed in gene expression. Our studies have revealed exciting and novel findings that elucidate the effects of EGFR signaling on the epigenetic landscape. Specifically, we have identified putative β-catenin targets that become activated following EGFR stimulation. We hypothesize that EGFR signaling promotes the activation of specific β-catenin genes in order to alter cellular identity. Methods: RNA-seq and ChIP-seq for H3K18ac and H3K27ac was conducted following an EGF treatment time course in SKBR3 cells. The levels of several proteins of interest were determined by Western blot analysis. The cellular localization of proteins of interest was examined using biochemically fractionated lysates followed by Western blot analysis. Results: RNA-seq analysis following an EGF treatment time course revealed that approximately 2,200 genes are either upregulated or downregulated compared to untreated cells. Moreover, the expression profiles clearly demonstrated waves of transcription. Next, we determined the status of H3K18ac and H3K27ac using ChIP-seq following an EGF time course. We found that H3K18ac and H3K27ac increased globally within 1h post-EGF treatment compared to untreated cells. We conducted a motif discovery search for transcription factor binding sites contained from -1000bp to +200bp for all activated genes and determined that each wave of transcription had some unique putative regulators. As expected, the genes activated at 1h and 2h post-EGF treatment contained c-Jun and JunD binding sites. Surprisingly, TCF3, TCF5, and LEF1 motifs were enriched in some genes that peaked in expression at 6h, 16h, and 24h post EGF treatment. Lastly, we biochemically fractionated the cellular compartments and detected an increase in chromatin associated β-catenin following EGF treatment, suggesting a crosstalk between EGFR and Wnt signaling components. We plan to determine the genome-wide localization of β-catenin following EGF treatment. Conclusions: Our data suggest that a crosstalk between EGFR and Wnt signaling components may regulate β-catenin target genes and lead HER2+ cells9 resistance to therapeutic treatment. Citation Format: Miguel Nava, Nwamaka Amobi, Nathan Zemke, Arnold Berk, Robin Farias-Eisner, Jay Vadgama, Yanyuan Wu. EGFR signaling mediated modulation of transcription and probable crosstalk with components of Wnt signaling [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr B68.

Abstract B36: A 1,5-diheteroarylpenta-1,4-dien-3-one inhibits TNBC cell growth by blocking AKT/mTOR/P70S6K pathway

One in 8 women will develop breast cancer during the course of her life. Recent developments in the field of drug development have provided a significant increase in progression free survival and decrease in mortality of this disease. However, one of the main problems in breast cancer remains the development of resistance to current treatments and relapse of the disease. Moreover, there is no known target for the treatment of the triple negative breast cancers (TNBC) subset, which is more aggressive than the other types of the disease. Therefore, it is imperative to develop new drugs that can block the activity of pro-survival signaling within the cancer cells and inhibit their growth, particularly in the TNBC subset. In an attempt to identify such drugs, we screened a panel of 13 compounds, containing the general structure of 1,5-diheteroarylpenta-1,4-dien-3-one (compounds 34 to 46), for their effectiveness in inhibiting the growth of both TNBC and ER+ breast cancers. The IC50s for these compounds were in the range of 63 to 8346 nM against various breast cancer cell lines. Interestingly, the most potent compound (# 34), (1E,4E)-1,5-bis(1-pentan-2-yl)-1H-imidazol-2-yl)penta-1,4-dien-3-one, showed its highest growth inhibitory activity in the TNBC cell line, MDA-MB-231, while also being effective in inhibiting the growth of ER+ cell lines to a lesser extent. Further investigations were then focused on this TNBC cell line. The mechanism of action of compound 34 was investigated by using a Human Phospho-Kinase Array, which revealed a significant decrease in p-P70S6K and p-mTOR. Additional assays confirmed the same results and also showed significant reductions in total mTOR and Total AKT as well as significant decreases in p-P70S6K and p-mTOR. Moreover, compound 34 treatment induced higher levels of p-c-JUN and p-P38, which may be indications of cell stress upon treatment. Overall our data indicate that compound 34 induces profound decreases in the activity of pro-survival pathways such as AKT/mTOR, which significantly inhibited growth of the TNBC cell line. Citation Format: Mohammad Atefi, Manee Patanapongpibul, Lynn Ma, Qiao-Hong Chen, Guangdi Wang, Yong Wu, Jay Vadgama. A 1,5-diheteroarylpenta-1,4-dien-3-one inhibits TNBC cell growth by blocking AKT/mTOR/P70S6K pathway [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr B36.

Abstract B33: Persistent activation of ERK and SMAD4 nuclear retention by high free fatty acids promote breast cancer metastasis

Obesity increases the risk of developing distant metastatic recurrence, and diminishes overall and breast cancer-specific survival; however, the mechanisms behind this correlation and relevant therapeutic targets are poorly defined. Plasma free fatty acids (FFA) levels are usually elevated in obese individuals. Here, using human breast cancer cell lines, we found that TGF-β transiently activates the extracellular signal-regulated kinase (ERK) and subsequently marks SMAD4 for phosphorylation, which facilitates SMAD4-ubiquitin specific peptidase 9, X-linked (USP9x) interaction, SMAD4 nuclear retention, SMAD3-SMAD4 complex nuclear accumulation, and stimulated TGF-β/SMAD3–mediated transcriptional activity of Twist and Snail. USP9x competitively inhibited the E3 ubiquitin-protein ligase transcriptional intermediary factor 1 γ (TIF1γ) from binding and monoubiquitinating SMAD4. This process maintained SMAD4 nuclear retention and stabilized the SMAD3/SMAD4 complex in the nucleus. Inhibition of ERK resulted in SMAD4 de-phosphorylation, USP9x-SMAD4 disassociation and SMAD4-TIF1γ interaction, which ultimately marks SMAD4 for mono-ubiquitination, leading to SMAD4 nuclear export and termination of the transcription of Twist and Snail. In addition, loss of USP9x abolished TGF-β–induced SMAD4 nuclear retention and SMAD3/SMAD4 formation, leading to inhibition of Twist and Snail expression. We also found that ERK-induced SMAD4 T277 phosphorylation and SMAD4-USP9x interaction was required for TGF-β–induced breast cancer invasion, which was attributed to SMAD3/SMAD4-dependent up-regulation of the transcription factors, Twist and Snail. Importantly, FFA further facilitates TGF-β–induced ERK activation, SMAD4 phosphorylation, SMAD4 nuclear retention and SMAD3/SMAD4 formation, thus promoting TGF-β–dependent cancer progression. Inhibition of ERK and USP9x suppressed obesity-induced metastasis. Additionally, phospho-ERK and -SMAD4 levels correlated with activated TGF-β signaling and metastasis in obese patient breast cancer specimens. Altogether, we demonstrated that USP9x promotes breast cancer invasion by promoting nuclear retention of SMAD4 and identified crosstalk between USP9x and TGF-β/ERK/SMAD4/SMAD3 pathways. Our study highlights the vital interaction of USP9x and SMAD4 for governing TGF-β signaling and dyslipidemia-induced aberrant TGF-β activation during breast cancer metastasis. Citation Format: Yong Wu, Xiaoting Yu, Kruttika Bhat, Sami Dwabe, Mohammad Atefi, Jay Vadgama. Persistent activation of ERK and SMAD4 nuclear retention by high free fatty acids promote breast cancer metastasis. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr B33.