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The abnormal PI3K/AKT/mTOR pathway is one of the most common genomic abnormalities in breast cancers including triple-negative breast cancer (TNBC), and pharmacologic inhibition of these aberrations has shown activity in TNBC patients. Here, we designed and identified a small-molecule Comp34 that suppresses both AKT and mTOR protein expression and exhibits robust cytotoxicity towards TNBC cells but not nontumorigenic normal breast epithelial cells. Mechanically, long noncoding RNA (lncRNA) AL354740.1-204 (also named as NUDT3-AS4) acts as a microRNA sponge to compete with AKT1/mTOR mRNAs for binding to miR-99s, leading to decrease in degradation of AKT1/mTOR mRNAs and subsequent increase in AKT1/mTOR protein expression. Inhibition of lncRNA-NUDT3-AS4 and suppression of the NUDT3-AS4/miR-99s association contribute to Comp34-affected biologic pathways. In addition, Comp34 alone is effective in cells with secondary resistance to rapamycin, the best-known inhibitor of mTOR, and displays a greater in vivo antitumor efficacy and lower toxicity than rapamycin in TNBC xenografted models. In conclusion, NUDT3-AS4 may play a proproliferative role in TNBC and be considered a relevant therapeutic target, and Comp34 presents promising activity as a single agent to inhibit TNBC through regulation of NUDT3-AS4 and miR-99s.

Human Papillomavirus (HPV), a non-enveloped epitheliotropic double stranded DNA virus, is an etiological agent of oral cancer, and can be cleared by host immunity. A small percentage of patients that develop persistent infection with oncogenic HPV show an increased risk of developing an HPV-associated malignancy. Head and neck, oropharyngeal cancer is related to the persistent infection by high-risk HPV type 16, E6/E7 oncoproteins. The E6/E7 oncoproteins significantly contribute to the carcinogenic genomic instability effect of high-risk HPV through the degradation of two proteins, p53 and pRB, respectively. The human oropharynx often harbors anaerobic bacteria that produce a variety of byproducts, including butyrate. Butyrate is a modulator of induced genomic instability, influencing the development of HPV oral cancer when implanted in a syngeneic mouse model. In this study, we investigate butyrate co-treatment with HPV E6/E7 in vitro. This treatment increased cellular proliferation, DNA double strand breaks, and genetic instability processes associated with apoptosis regulation. These data indicate that in connection with butyrate, HPV E6/E7 oncoproteins can promote increased upregulation of apoptosis, disrupt cell cycle regulation, and decrease DNA double strand break repair. Epigenetics and chromosomal instability events have been noted, specifically, the deletion of the distal region of chromosome 11q22-23 in E6/E7 transfected keratinocytes. These results indicate that HPV-16, E6/E7 oncoproteins plus butyrate treatment can influence genomic changes in mouse keratinocytes, which may further promote oral carcinogenesis. Citation Format: Eva McGhee, Mengtao Li, Yi-Ling Lin, Brooke Su-Velez, Nefertari Carr, Chinelo Njubigbo, Rohun Sadeghi, Matin Sadegh, Julian Handler, Adin Handler, Naomi Long, Jay Vadgama. Exposure to butyrate metabolite mediates genomic instability in oropharyngeal keratinocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1510.

BACKGROUND: Endometriosis is complex, but identifying the novel biomarkers, inflammatory molecules, and genetic links holds the key to the enhanced detection, prediction and treatment of both endometriosis and endometriosis related malignant neoplasia. Here we review the literature relating to the specific molecular mechanism(s) mediating tumorigenesis arising within endometriosis. METHODS: Guidance (e.g. Cochrane) and published studies were identified. The Published studies were identified through PubMed using the systematic review methods filter, and the authors’ topic knowledge. These data were reviewed to identify key and relevant articles to create a comprehensive review article to explore the molecular fingerprint associated with in endometriosis-driven tumorigenesis. RESULTS: An important focus is the link between C3aR1, PGR, ER1, SOX-17 and other relevant gene expression profiles and endometriosis-driven tumorigenesis. Further studies should also focus on the combined use of CA-125 with HE-4, and the role for OVA1/MIA as clinically relevant diagnostic biomarkers in the prediction of endometriosis-driven tumorigenesis. CONCLUSIONS: Elucidating endometriosis’ molecular fingerprint is to understand the molecular mechanisms that drive the endometriosis-associated malignant phenotype. A better understanding of the predictive roles of these genes and the value of the biomarker proteins will allow for the derivation of unique molecular treatment algorithms to better serve our patients.

A greater understanding of factors causing cancer initiation, progression and evolution is of paramount importance. Among them, the serine/threonine phosphatase PPM1D, also referred to as wild-type p53-induced phosphatase 1 (Wip1) or protein phosphatase 2C delta (PP2Cδ), is emerging as an important oncoprotein due to its negative regulation on a number of crucial cancer suppressor pathways. Initially identified as a p53-regulated gene, PPM1D has been afterwards found amplified and more recently mutated in many human cancers such as breast cancer. The latest progress in this field further reveals that selective inhibition of PPM1D to delay tumor onset or reduce tumor burden represents a promising anti-cancer strategy. Here, we review the advances in the studies of the PPM1D activity and its relevance to various cancers, and recent progress in development of PPM1D inhibitors and discuss their potential application in cancer therapy. Consecutive research on PPM1D and its relationship with cancer is essential, as it ultimately contributes to the etiology and treatment of cancer.

A growing body of literature supports the role of apolipoproteins present in HDL in the treatment of pro-inflammatory diseases including cancer. We examined whether bovine HDL (bHDL) and three dual-domain peptides, namely AEM-28 and its analog AEM-28-2, and HM-10/10, affect tumor growth and development in mouse models of ovarian and colon cancer. We demonstrate that bHDL inhibits mouse colorectal cancer cell line CT26-mediated lung tumor development, and mouse ovarian cancer cell line ID8-mediated tumor burden. We also demonstrate that, although to different degrees, dual-domain peptides inhibit cell viability of mouse and human ovarian and colon cancer cell lines, but not that of normal human colonic epithelial cells or NIH3T3 mouse fibroblasts. Dual-domain peptides administered subcutaneously or in a chow diet decrease CT26 cell-mediated tumor burden, tumor growth, and tumor dissemination in BALB/c mice. Plasma levels of lysophosphatidic acid (LPA) are significantly reduced in mice that received bHDL and the dual-domain peptides, suggesting that reduction by effecting accumulation and/or synthesis of pro-inflammatory lipids may be one of the mechanisms for the inhibition of tumor development by bHDL and the dual-domain peptides. Our studies suggest that therapeutics based on apolipoproteins present in HDL may be novel agents for the treatment of epithelial adenocarcinomas of the ovary and colon.

Aberrant PP2Cδ inhibits p300-mediated p53 acetylation and activation via the ATM/BRCA1 pathway, leading to cancer progression., Although nuclear type 2C protein phosphatase (PP2Cδ) has been demonstrated to be pro-oncogenic with an important role in tumorigenesis, the underlying mechanisms that link aberrant PP2Cδ levels with cancer development remain elusive. Here, we found that aberrant PP2Cδ activity decreases p53 acetylation and its transcriptional activity and suppresses doxorubicin-induced cell apoptosis. Mechanistically, we show that BRCA1 facilitates p300-mediated p53 acetylation by complexing with these two proteins and that S1423/1524 phosphorylation is indispensable for this regulatory process. PP2Cδ, via dephosphorylation of ATM, suppresses DNA damage–induced BRCA1 phosphorylation, leading to inhibition of p300-mediated p53 acetylation. Furthermore, PP2Cδ levels correlate with histological grade and are inversely associated with BRCA1 phosphorylation and p53 acetylation in breast cancer specimens. C23, our newly developed PP2Cδ inhibitor, promotes the anticancer effect of doxorubicin in MCF-7 xenograft–bearing nude mice. Together, our data indicate that PP2Cδ impairs p53 acetylation and DNA damage response by compromising BRCA1 function.

Coronavirus disease-19 (COVID-19). Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a novel coronavirus resulting in a severe threat to safety and public health. Genetic analyses suggest that SARS-COV-2 belongs to the Betacoronavirus group and Middle East respiratory syndrome coronavirus (MERS-CoV). Like most viruses, some are oncogenic, and genetic instability is involved in the initiating factor for tumor progression that is known to lead to malignancy, resulting in disease sequelae. Genomic instability is characterized by the increased rate of acquisition of alterations in the mammalian genome. These changes encompass a diverse set of biological end points including gene mutation and amplification, cellular transformation, clonal heterogeneity and delayed reproductive cell death. The loss of stability of the genome is becoming accepted as one of the most important aspects of viral induced carcinogenesis, and the numerous genetic changes associated with cancer cell implicate genomic stability as contributing to the neoplastic phenotype. The aim of this study is to analyze the influence of COVID-19 cancer related diseases and determine the epigenetic-genomic profiles in these diseases. In our experimental procedures, we will use oral epithelium cells as a model for this cancer study. We proposed a novel approach to study COVID-19-induced genetic changes with oral epithelium cells. We used these cells with COVID-19 co treatment to determine mechanisms that lead cells into crisis. Preliminary data using oral viral infected cells show that the viral infection can trigger certain gatekeeper proteins like p53 and pRB to be degraded and forcing cells to go into crises, and telomerase is turned on. In this study we will determine if COVID-19 transfected cells can follow the process using the Hayflick model, which show cells that are in crises, and some may progress to cancer. In summary, preliminary data show that viral infected cells that are none cancerous and are telomerase positive, may use a similar mechanism to send cells into crisis that turn telomerase on resulting in cellular malignancy. Citation Format: Eva McGhee, Mengtao LI, Yi-Ling Lin, Nefertari Carr, Chinelo Njubigbo, Rohun Sadeghi, Brooke Su-Velez, Julian Handler, Adin Handler, Judith Okoro, Sebhat Afework, Jay Vadgama. The hallmark of COVID-19 cancer disparities: Cellular sequelae [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 722.

Ovarian cancer (OC) is a serious condition that often presents at advanced stages and has high mortality rates, with the current mode of early-stage screening lacking sensitivity and specificity. OC often presents asymptomatically, which renders early diagnosis difficult. Furthermore, many patients lack significant risk factors or family history of the disease. Five-year survival rates differ between patients with OC among racial, ethnic, and social groups as a result of different social barriers. This review article aims to present the currently existing data regarding health care disparities among OC patients of different ethnic, demographic, and socioeconomic backgrounds, and what next steps should be taken to better understand and eventually eliminate these potentially devastating health care disparities. Increasing data support the notion that a combination of genomic, socioeconomic status, social factors, and cultural differences lead to differential treatments and therefore health care disparities. While genomic and biological factors are important, language barriers, geographic and travel barriers, differences in comorbidity likelihood between populations, and different treatment plans seem to have a greater impact on 5-year survival rates of patients from diverse backgrounds. Language barriers limit a shared-decision model of care. Transportation limitations and geographic differences can lead to limited follow-up and insufficient care in resource and equipment restrictive settings. Patients with these barriers also tend to have a higher incidence of comorbidities that raise the mortality rate of OC. Further research needs to explore effective solutions to bridge health care disparities and understand why they occur.

The human papillomavirus (HPV) causes 70% of oropharyngeal cancers in the United States. While the molecular and cellular mechanisms and the stepwise progression of HPV-related cervical cancer have been well studied, oropharyngeal HPV carcinogenesis remains to be unraveled since an equivalent HPV premalignant stage has never been identified in clinical oropharyngeal tissues. In this study, a series of keratinocyte cell lines derived from the base of the tongue of C3H mice were established. These cells formed varying degrees of malignancy in the syngenetic mice and are used to study the biology of HPV-related oropharyngeal cancer in an immunocompetent host. Keratinocytes from the base of the tongue were isolated from C3H newborns and were subsequently retrovirally immortalized with TERT or HPV16 E6E7 oncoproteins, with or without oncogene H-ras. They are designated MOKT1 (TERT), MOKT1/H-ras (TERT/H-ras), MOKE2 (E6E7), or MOKE2/H-ras (E6E7/H-ras). These cells were injected in the masseter muscle of syngeneic mice and the developing lesions were harvested for histological analysis over a time course of 1 month. E6E7 transformed MOKE2 cells exhibited features of HPV associated cancer cells. We found that p63 and phosphorylated STAT3 were upregulated in MOKE2 cells in comparison to MOKT1 cells. MOKE2 cells obtained growth advantages with increased cell proliferation and decreased apoptosis. In addition, MOKE2 cells showed increased cell migration activity in wound healing assays. While all cell lines grew indefinitely in vitro, MOKT1 and MOKT1/H-ras failed to show any sign of malignancy when implanted in vivo. MOKE2 demonstrated features of malignancy, but the phenotype was transient. Host immunity does not appear to be the determining factor for suppressing tumor growth as MOKE2 regressing also occurred when implanted in immunodeficient NSG mice. Only MOKE2/H-ras cells were capable of progressing and establishing permanent cancer. Ourdata suggest that differentiation induced cell growth arrest may contribute to tumor regression and a second hit is required to overcome such arrest. The MOKE2 cells can be used in syngeneic mice to study secondary oncogenic factors in the development of HPV-related oropharyngeal cancer. Citation Format: Eva McGhee, Yi-Ling Lin, Jay Vadgama, Khadijah Lang, Judith Okoro, Victoria Vidal, Naomi Long, Mengtao Li. A mouse model for studying oncogenesis of HPV-associated oropharyngeal cancer [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 427.

Malignant progression of high-risk Papillomavirus (HPV), associated lesions is a slow and inefficient process. HPV a non-enveloped epitheliotropic double stranded DNA virus, is an etiological agent of oral cancer. Oral cancer is related to the persistent infection by high-risk HPV type 16, E6/E7 oncoproteins. The E6/E7 complex significantly contribute to the carcinogenic genetic instability effect of high-risk HPV through the degradation of two gatekeeper proteins, p53 and pRB, respectively. This process is associated with viral genome integration into a host cellular chromosome and accumulation of numerical and structural chromosome aberrations. Viral genome integration results in consistent, sustained and dysregulated expression of the E6 and E7 oncogenes, and continued E6/E7 expression is necessary for maintenance of the transformed phenotype of HPV positive cancer cell. Induction of genomic instability by high-risk HPV E6/E7 demonstrates that HPV also drive malignant progression through targeting the nuclear and mitotic apparatus protein-1 (NuMA), and through subversion of DNA double strand break repair by HPV E6/E7 induced centrosome abnormalities. This in turn may cause abnormal localization of the microtubule protein dynein. The NuMA provides a cohesive force that guarantees the integrity of the mitotic spindle poles. However, there are no studies addressing the relationship between HPV16 infection-oral cancer, NuMA expression, and genomic instability. The aim of this study determined if HPV16 E6/E7 subverts centrosome coalescence in oral cancer caused by HPV oncogenes targeting the NuMA protein. In our experimental procedures, we proposed a novel approach to study HPV-induced chromosomal changes with reconstructed human oral epithelium in SCID mouse. We used HPV16, which is the most common HPV type found in oral cancer. Two plasmids were used to produce HPV-16 E6/E7. The first plasmid p16sheLL expresses the two viral capsid proteins, L1 and L2. The second plasmid pBR322HPV16 contains the full length HPV-16 genome. This approach closely mimics the architecture of normal human oral epithelium. In our study, we first focused on analyzing selected cellular processes involving multipolar mitoses. We examined Dynein localization in mitotic cells by immunofluorescence. We also analyzed progressive morphological and cytopathic changes in HPV-infected oral epithelia by using molecular cytogenetic analysis. In summary these data indicate that NuMA expression is alternated, and HPV16 E6/E7 oncoproteins can promote increased upregulation of genomic instability by induction of centrosome abnormalities and inhibition of DNA double strand break repair. We also found High-risk HPV16 E6/E7 oncoproteins induced mitotic defects and genetic aneuploidies through induction of supernumerary anaphase bridges, supernumerary centrosomes, and DNA double strand breaks. Citation Format: Eva McGhee, Mengtao Li, Yi-Ling Lin, Khadijah Lang, Meidrah Tyler, Judith Okoro, Mai Do, Naomi Long, Jenna Cormier, Adin Handler, Julian Handler, Sameeran Das, Benjamin Liu, Anthonia Duru, Billy Ballard, Roland Pattillo, Jay Vadgama. Genomic instability changes acquired by HPV16 E6/E7 targeting multipolar mitoses aneuploidy: Cellular sequelae [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 3789.

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.

Epidemiological data demonstrate that patients with diabetes have an augmented risk of developing various types of cancers, accompanied by higher mortality. A number of mechanisms for this connection have been hypothesized, such as insulin resistance, hyperinsulinemia, hyperglycemia, and increased inflammatory processes. Apart from these potential mechanisms, several diabetes-associated dysregulated cytokines might be implicated in the link between diabetes and cancer. In fact, some inflammatory cytokines, e.g. TNF-α, IL-6 and leptin, have been revealed to play important roles in both initiation and progression of tumor. Here, we depict the role of these cytokines in key events of carcinogenesis and cancer development, including their capability to induce oxidative stress, inflammation, their participation in epithelial mesenchymal transition (EMT), angiogenesis, and metastasis. Finally, we will also highlight the existing knowledge in terms of the involvement of these cytokines in different cancer types and comment on potential significances for future clinical applications.

Introduction: Human Epidermal Growth Factor Receptor-2-positive (HER2+) breast cancer is one of the four major molecular types of breast cancer. HER2 is a classical receptor tyrosine kinase (RTK) and its kinase activity is stimulated by heterodimerization with other ligand bound HER family members, such as EGFR/HER1. Treatment for HER2+ breast cancer includes the use of Trastuzumab, a monoclonal antibody that binds to the HER2 extracellular domain and inhibits downstream signaling. Resistance to Trastuzumab involves several cellular and molecular alterations, including gain of function mutations in proteins that are downstream of HER2 activation. The purpose of this study is to examine EGFR signaling in HER2+ breast cancer cells to discover novel putative drug targets. Methods: HER2-positive breast cancer cell line (SKBR3) was used before and after EGF treatment. In order to determine whether EGFR signaling was activated, the levels of several proteins of interest were determined by western blot analysis. RNA-seq and ChIP-seq for H3K18ac and H3K27ac was conducted following an EGF time course in SKBR3 cells. Results: Treatment of SKBR3 cells with EGF resulted in a transient increase in pERK1/2 and pAKT, which returned to basal levels at 1h and 2h post-EGF treatment, respectively. This was surprising because pEGFR remained higher in treated cells compared to untreated cells throughout the 24h time course. Immediate early genes (IEGs) became activated in SKBR3 cells, as previously reported in other cell types, during the early phase of EGFR stimulation ( Citation Format: Miguel Nava, Nathan R. Zemke, Arnie Berk, Robin Farias-Eisner, Jay Vadgama, Yanyuan Wu. EGFR signaling activates S100 gene expression in HER2-positive breast cancer cells [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 362.

Human Papillomavirus (HPV), a non-enveloped epitheliotropic double stranded DNA virus, is an etiological agent of oral cancer. Oral cancer is related to the persistent infection by high-risk HPV type 16, E6/E7 oncoproteins. The E6/E7 oncoproteins significantly contribute to the carcinogenic genetic instability effect of high-risk HPV through the degradation of two gatekeeper proteins, p53 and pRB, respectively. The ATM gene, also known as a gatekeeper protein plays a central role in the complex processes that repair DNA strand breaks. The ATM gene is located at 11q22.3 and belongs to a protein family known as the PI3K. Many studies have tried to clarify the epigenetic instability role of ATM in cancer function and susceptibility. However, there are no studies addressing the relationship between HPV infection-oral cancer, ATM/PI3K expression, and genomic instability. The aim of this study is to analyze the influence of HPV16 E6/E7 oncoproteins in oral cancer and the epigenetic profile. In our experimental procedures, we proposed a novel approach to study HPV-induced epigenetic changes with reconstructed human oral epithelium in SCID mouse. We used HPV-16, which is the most common HPV type found in oral cancer. Two plasmids were used to produce HPV-16 E6/E7. The first plasmid p16sheLL expresses the two viral capsid proteins, L1 and L2. The second plasmid pBR322HPV16 contains the full length HPV-16 genome. This approach closely mimics the architecture of normal human oral epithelium. In our study, we first focused on analyzing selected cellular gene loci where DNA methylation profiles are known to be altered, either by HPV infection or in oral cancer. We examined the following gene promoters DcR1/DcR2 (apoptosis regulation), p16 (cell cycle control), DAPK (cancer metastasis), and MGMT (DNA repair). We also analyzed progressive morphological and cytopathic changes in the HPV-infected oral epithelia. These data indicate that HPV E6/E7 oncoproteins can promote increased upregulation of apoptosis, disrupt cell cycle regulation, and decrease DNA double strand break repair. Epigenetics and chromosomal instability events were noted, specifically, the deletion of the distal region of chromosome 11q22-23 in the E6/E7 transfected keratinocytes. These results indicate that HPV-16, E6/E7 oncoproteins induced epigenetic changes in mouse keratinocytes, which may further promote oral carcinogenesis. Citation Format: Eva McGhee, Mengtao Li, Yi-Ling Lin, Liliana Zarate, Naomi Long, Mai Do, Chinelo Ezechukwu, Nichelle Cox, Hyun Chung, Jenna Cormier, Meidrah Tyler, Victoria Vidal, Billy Ballard, Roland Pattillo, Jay Vadgama. Upregulation of epigenetic changes acquired by HPV16 E6/E7 oncoproteins in mouse keratinocytes: Targeting ATM/PI3K [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 3326.

Human papillomavirus (HPV), high-risk type 18, is directly associated with approximately 98% incidence of invasive cervical cancer. Epithelial cells infected with HPV-18 become transformed and exhibit overexpression of telomerase activity and chromosomal instability. As a result of this transformation, E6 and E7 oncoproteins are perpetually expressed: E6 degrades the tumor suppressor p53; E7 inhibits the tumor suppressor pRB, leading to uncontrollable growth and the extension of telomere length. While a few studies explored the chromosomal instability induced by HPV E6 and E7 oncoproteins, the full scope of the problem has not been clearly characterized. In this research we investigated HPV-18 acquired genomic instability by E6 and E7 oncoproteins. HPV-positive cervical cancer cells, HeLa, were studied through spectral karyotyping (SKY), Giemsa banding (G-banding), telomere length, and telomerase activity using PCR ELISA assay and the TRAPeze XL Kit, which uses fluorescence energy transfer. With the use of SKY, G-banding, and chromosomal instability analysis, HeLa cells exhibited many aneuploidies. In our results, there were consistent translocations on chromosomes 4 and 11, with deletions on chromosomes 11 and 20. There were also other translocations on chromosomes 9 and 20, with deletions on chromosome 10. Additionally, copies of chromosome 5 and unidentifiable marker chromosomes were noted. Moreover, telomerase data suggest that upregulated expression of telomerase activity correlates with the increase in chromosomal instability. The aforementioned aneuploidy is demonstrative of the induced chromosomal instability from HPV-18 infection. In addition to the tumor-suppressor disruption acquired by E6 and E7 oncoproteins, HeLa cells showed gene deletion on chromosome 11. The ATM gene, which is specifically located between distal regions 11q22.3 and 11q23 of chromosome 11, is known to help identify breaks in DNA and plays a crucial role in DNA repair. Damage to the ATM gene may further play an important role in increasing cancer progression. Citation Format: Mai Do, Nichelle Cox, Naomi Long, Liliana Zarate, Chinelo Ezechukwu, Jenna Cormier, Hyun Chung, Meidrah Tyler, Judith Okoro, Diondra Harris, Victoria Vidal, Jerica Watson, Ellie Canty, Shyam Arya, Benjamin Liu, Roland Pattillo, Billy Ballard, Jay Vadgama, Eva McGhee. Molecular characterization of HPV type 18 cervical cancer: Upregulation of telomerase expression and induced chromosomal instability by E6 and E7 oncoproteins [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 3388.

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.

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.

Background: Experimental evidence supports a protective role of Vitamin D level in breast carcinogenesis, but epidemiologic evidence is inconsistent. African Americans have high prevalence of Vitamin D deficiency and the African American women with breast cancer have the highest mortality rate. The aim of this study is to investigate the association of Vitamin D levels with breast cancer risk and disease progression in African American and Hispanic women. Methods: This is a cases-control study of 660 African American and Hispanic women with and without breast cancer in South Los Angeles. Blood samples were collected prior cancer treatment and the serum 25-hydroxyvitamin D (25(OH) D was measured by Quest Diagnostics. Information regarding BMI and clinical factors were obtained by medical record abstraction. Logistic Regression with multivariate analysis was used for determining the association of Vitamin D deficiency ( Results: Our data shows that 69.2% of African American women and 37.8% of Hispanic women suffer from Vitamin D deficiency in our cohort. African American women had significant lower level of Vitamin D3 compared with Hispanic women in all age groups. The lower Vitamin D3 level was observed among age groups of 31 to 50 in African Americans. The deficiency in Vitamin D levels was significantly associated with breast cancer in both African Americans (OR=2.5, p=0.007) and Hispanics (OR=1.9, p=0.009). Interestingly, we found that a significant association of Vitamin D deficiency with trip negative breast cancer (TNBC) in African Americans (OR=3.2, p=0.04), but not in Hispanics. The lowest Vitamin D3 level was observed in African American women with TNBC. There was no significant association of Vitamin D deficiency with tumor size, lymph node involvement and tumor stage. The deficiency of Vitamin D3 level was not associated with disease progression in this cohort of women. Conclusion: Our data suggests that a significant association of Vitamin D deficiency with breast cancer in both African Americans and Hispanics, especially more associated with type of TNBC in African American women in our cohort. Citation Format: Yanyuan Wu, Marianna Sarkissyan, Sheilah Clayton, Jay Vadgama. Association of Vitamin D deficiency with breast cancer in African-American and Hispanic women in south Los Angeles [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 5279. doi:10.1158/1538-7445.AM2017-5279

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.

The association of human papillomavirus (HPV) with cervical cancer, head and neck cancer is well established, but the involvement of HPV E6/E7 oncoproteins in breast cancer is more contentious. Previous studies suggest that high risk HPV E6/E7 oncoproteins inactivate two tumor suppressor proteins p53 and pRb signaling and increase cell proliferation. There is cumulative evidence that high risk HPV 16 E6/E7 oncoproteins may have causal roles in some human breast cancers. Moreover, some studies have identified HPV DNA in breast tissue and breast cancer specimens. Nevertheless, the route of transmission of HPV in breast cells has not been determined, and the mechanisms by which HPV involvement in breast cancer are not known. We used a non-tumorigenic (MCF12A) epithelial breast cell line transfected with HPV 16 to elucidate the role of oncoproteins E6/E7 in pro-proliferative and anti-apoptotic signaling pathways. We also used Tamoxifen treatment, which is an antagonist of the estrogen receptor in breast tissues through its active metabolite, 4hydroxytamoxifen. We demonstrate that HPV E6/E7 transfection activates the extracellular signal-regulated kinase (ERK) MAPK pathway and Akt/PKB kinase signaling pathways through activating estrogen receptor alpha in MCF12A cells. E6/E7 oncoproteins transfection alone dramatically increases estrogen receptor alpha protein expression. Treatment with tamoxifen, an antagonist of the ER, significantly abolishes E6/E7-increase in ERK and PKB phosphorylation without altering their protein levels, suggesting that the estrogen receptor is a key mediator of HPV E6/E7 mediated ERK and Akt/PKB activation. Our current work show an insightful understanding of the molecular mechanisms of HPV-elicited Akt/PKB activation and its roles in cell proliferation and survival in normal breast epithelial cells. Therefore, these findings indicate that HPV E6/E7 oncoproteins may promote the transforming activities of high-risk HPV E6/E7 proteins and cell proliferation in breast cells. Therefore, suppression of estrogen receptor signaling networks may be used as a therapeutic strategy for HPV associated lesions and cancers such as breast metastasis. Citation Format: Eva McGhee, Lucy Tran, Mengtao Li, Yong Wu, Seyung Chung, Cheryl Araniego, Karen Tate, Melanie Baker, Kamilah Evans, Mechelle Rouse, Jay Vadgama. Estrogen receptor alpha mediates HPV E6/E7 oncoproteins: Induced breast cells proliferation. [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 4588.

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 (CTGF or CCN2) is a secreted protein that belongs to the CCN [cysteine-rich CYR61/connective tissue growth factor/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 the present 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 anti-apoptotic proteins, Bcl-xl, Survivin, and Flip. Over-expression of CTGF caused the U343 GBM cells to survive for longer than 40 days in serum-free medium and resist anti-tumor drugs including tumor necrosis factor (TNF), TNF-related apoptosis-inducing ligand (TRAIL), 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.

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.

Experiments were conducted to gain insight into calcium signaling mechanisms triggered by angiotensin II (AngII) stimulation in vascular smooth muscle cells (SMC) freshly isolated from preglomerular vessels of normotensive Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Cytosolic calcium concentration ([Ca2+]i) was measured using ratiometric Fura-2 fluorescence and a microscope-based photometer. Vascular SMC from preglomerular vessels were isolated and dispersed using an iron oxide-sieving method combined with collagenase treatment. AngII produced rapid increases in [Ca2+]i that remained elevated for the duration of continued stimulation. The same pattern of time response was observed in WKY and in SHR. AngII elicited dose-dependent increases in [Ca2+]i in groups of individual preglomerular arteriolar SMC from both strains. AngII (10(-10) M) induced an increase from baseline levels in WKY and SHR (37+/-9 and 32+/-13 nM; P 0.4). The effects of AngII were inhibited by 88% by the AT1 receptor blocker candesartan in renal SMC. In SMC pretreated with calcium-free medium, baseline [Ca2+]i fell by about 60 nM. Thereafter, AngII did not elicit any [Ca2+]i response either in WKY or in SHR when calcium entry was prevented. Also, after prestimulation by AngII, a calcium-free solution completely reversed the effects of AngII. This study shows that AngII acts through AT1 receptors to stimulate [Ca2+]i by a predominant action on calcium entry with no evidence for calcium mobilization. Other studies have demonstrated that calcium entry in these SMC is mediated by voltage-gated, L-type entry channels sensitive to dihydropyridine agents. No strain differences were noted between the actions of AngII on individual renal SMC from SHR and normotensive control animals.

Hyperlipidemia is an established risk factor for progressive renal damage and proteinuria. Platelets and vascular smooth muscle cells (VSMC) are known to possess low density lipoprotein (LDL) cholesterol receptors. We used platelet LDL receptors to investigate the hypothesis that elevated lipids could activate intracellular calcium [Ca2+]i signals, leading to altered vascular tone and permeability. We divided essential hypertensives into microalbuminuric positive (MA+) and negative (MA-) groups and measured baseline and LDL stimulated levels of [Ca2+]i. The MA+ group demonstrated a significantly higher mean baseline [Ca2+]i level (119.0 +/-24.5 v 86.2 +/- 25.4 micromol/mL, P = .001). The MA+ group also displayed greater elevations in [Ca2+]i levels after stimulation with LDL in concentrations of 10 and 25 microg/mL (100.9 +/- 54.8 v 40.9 +/- 20.2, P = .04 and 111.6 +/- 51.0 v 52.9 +/- 39.9 micromol/mL, P = .03, respectively). Our data indicate that hypertensive patients with early glomerular capillary injury display exaggerated influx of [Ca2+]i after LDL receptor stimulation. Heightened LDL receptor sensitivity may facilitate LDL mediated [Ca2+]i signals, leading to increased VSMC tone and proliferation and progressive renal disease.

Background Phosphodiesterase 4D (PDE4D) degrades the secondary messenger adenosine 3’, 5’-cyclic monophosphate (cAMP). PDE4D recently was implicated in cancer cell proliferation, but alterations in its genome and function in tumors are unclear. Methods PDE4D gene copy number determined by SNP-CHIP was comprehensively analyzed in a large number of solid tumor specimens from diverse origins (n=5,569). Expression of PDE4D gene transcripts were examined in 971 primary tumors, as well as established cancer cell lines. PDE4D protein expression in 11 different types of cancers were evaluated by immunohistochemistry (n=165). Endogenous PDE4D in multiple types of tumor cell lines was suppressed by both shRNAs and compound 26B, a novel inhibitor of PDE4D, and the effects on apoptosis and proliferation were examined. We further investigated the molecular mechanisms involved. Effect of ectopic expression of PDE4D on tumor growth was assessed both in vitro and in vivo. Results Homozygous deletion of PDE4D occurred in 198 cases out of 5,569 primary solid tumors (3.56%), with most of them being internal microdeletions. Unexpectedly, these internal microdeletions did not result in loss of its gene products, as the expression of PDE4D mRNA and protein was not compromised in the PDE4D-microdeleted samples. Immunohistochemistry staining revealed that PDE4D protein levels were up-regulated in several types of tumors. Importantly, genetic and pharmacological suppression of endogenous PDE4D caused apoptosis and growth inhibition preferentially in a variety of cancer cells but not in non-malignant epithelial cells. We further showed that anti-tumor events triggered by PDE4D-suppression were lineage-dependently associated with induction of BIM and down-regulation of MITF. Moreover, ectopic expression of the PDE4D short isoform (PDE4D2) enhanced the proliferation of melanoma both in vitro and in vivo. Conclusions We show for the first time that although targeted by genomic homozygous microdeletions, alternatively spliced PDE4D can function as a tumor-promoting factor. PDE4D represents a novel, targetable enzyme of cancer cells. Citation Format: Dechen Lin, Liang Xu, Ling-Wen Ding, Arjun Sharma, Li-Zhen Liu, Henry Yang, Patrick Tan, Jay Vadgama, Beth Karlan, Jenny Lester, Nicole Urban, Michèl Schummer, Ngan Doan, Jonathan Said, Martin Walsh, Paresma Patel, Craig Thomas, Daniel Chan, Phillip Koeffler. Genomic and functional characterizations of phosphodiesterase 4Din human cancers. [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 586. doi:10.1158/1538-7445.AM2013-586

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

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

Background: Obesity is considered a major risk factor for cancers, in particular breast cancer. This phenomenon is becoming a major public health concern. Hence, modifying life-styles that could reduce obesity offers the potential for prevention as well as improvement of outcomes from cancer. Though the exact mechanisms of the effects of obesity on the etiology and progression of breast cancer are yet unclear, some studies suggest that increased body fat is associated with increased levels of estrogen and growth factors such as insulin-like growth factor 1 (IGF-I) which promote tumorogenesis. The effects of obesity and breast cancer in African-American women and Latina women have been explored in a limited number of studies. The aim of this study is to investigate the association of obesity with breast cancer in our cohort of minority women. Methods: Our study is a hospital based comparative study of over 300 African-American and Latina women in South Los Angeles who visited the Mammography Screening unit and/or the Hematology Oncology Clinic at Martin Luther King Ambulatory Care Center (MACC, formerly known as King-Drew Medical Center). Information regarding BMI and clinical and lifestyle-factors were obtained by medical record abstraction. Logistic regression with multivariate analysis was used to determine the association between obesity, breast cancer, and additional variables. We also examined the effect of comorbidity on breast cancer. Results: Our results show a significant association of breast cancer with obesity. Women with breast cancer were more likely to be obese (BMI>30) than women without breast cancer (OR =1.33; P-value Conclusion: Our data suggest an association of obesity with breast cancer, especially in premenopausal women and most significantly in the pre-menopausal African-American cohort. In addition, we show that hypertension as comorbidity plays a significant role in for breast cancer. Citation Information: Cancer Epidemiol Biomarkers Prev 2010;19(10 Suppl):B72.

Introduction: Estrogen receptor positive breast tumors frequently become resistant to Tamoxifen therapy. Elucidating mechanisms that contribute to resistance are critical for developing therapeutic targets. Previously, we have shown in our lab that FOXO1, a pro-apoptotic transcription factor, plays a role in Trastuzumab resistance, and is inactivated in Her2-overexpressing tumors associated with increased AKT activation. Restoration of FOXO1, both by inhibiting the AKT pathway and by FOXO1 transfection, restored cell-sensitivity to Trastuzumab. Currently, we are exploring whether FOXO1 may play a similar role in resistance to Tamoxifen in ER positive breast cancer cells. We hypothesize that loss of function of FOXO1 may contribute to Tamoxifen resistance and reactivation may restore sensitivity to tamoxifen. Methods: We have generated two tamoxifen resistant clones and in addition have studied a Tamoxifen resistant clone called MCF-7 TAMR/HER2 (HER2-transfected). tamoxifen resistant clones were generated by passaging wildtype MCF-7 (WT) cells through sphere cultures in the presence of tamoxifen. These clones are termed MCF-7 TAMR7 and MCF-7 TAMR26, representing 7 and 26 passages in sphere culture respectively. Cell line characteristics were assayed by immunohistochemistry (IHC) and RT-Q-PCR. Furthermore, MTT assays were used to test for E2 response in WT and TAMR cells. Results: Tamoxifen resistance was acquired by utilizing the sphere assay in the presence of Tamoxifen. IHC results revealed a significant loss of ER receptor in TAMR7 and TAMR26 clones. RT-Q-PCR demonstrated 85% loss of ER receptor mRNA in the TAMR7 and TAMR26 compared to WT cells. There was no difference in ER expression in WT and TAMR/HER2 cells. KI67 and CyclinD1 expression were lower in TAMR (TAMR7 and TAMR26) cells compared to WT and TAMR/HER2 cells. TAMR26 cells had lower proliferative response to E2 compared to WT cells. There was increased expression of CK5 in TAMR26 cells. FOXO1 was phosphorylated and located in the cytoplasm of the TAMR cells (TAMR7 and TAMR26) compared to WT and TAMR/HER2 cells. Conclusions: This study demonstrates that one mechanism of Tamoxifen resistance may be through the loss of the Tamoxifen target, the estrogen receptor (ER). TAMR clones isolated from mammosphere cultures with long term Tamoxifen treatment acquire an ER negative phenotype. These cells demonstrate loss of function of FOXO1. In addition, TAMR cells with high HER2 and normal ER, also acquire Tamoxifen resistance, by mechanisms not clearly understood. Further studies are currently underway in our lab to explore these mechanisms. Citation Information: Cancer Epidemiol Biomarkers Prev 2010;19(10 Suppl):A104.