Article Database

Abstract B15: BRCA haploinsufficiency promotes gluconeogenesis in fallopian tube epithelial cells

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Introduction: High-grade serous ovarian cancer (HGSC) is one of the deadliest gynecologic malignancies due to presentation at advanced stages and the development of chemotherapy resistance. The recent discovery that HGSC arises from fallopian tube epithelia (FTE) and the analysis results obtained from TCGA study are helping in understanding the genes involved in the progression of the disease. HGSC is genetically unstable due to the loss of function mutations in BRCA1, BRCA2, PALB2, ATM, and RAD51 genes involved in DNA damage response. These genes also regulate cellular metabolism, antioxidant, and detoxification responses. Preliminary data show that phase II antioxidant enzymes are significantly reduced in HGSC and there is an increase in reactive oxygen species (ROS) in cells harboring p53 and BRCA1 mutations in precursor lesions of FTE and HGSC. We also observed differential mRNA expression of PCK1 and PCK2, GLUT1 genes involved in gluconeogenesis and glycolysis. Mitochondria are one of the major organelles for the production of ROS, and it is a well-known fact that metabolism is drastically changed in cancer cells compared to normal cells. Our aim was to determine the metabolic and fuel dependency in preneoplastic transformed cells, characterize the mitochondrial phenotype, and link these dependencies to transformation in FTE.

Methods: Fallopian tube specimens were obtained from the Biospecimen Shared Resources Core at SCCC. Patients undergoing risk reduction surgery were consent and FTE cells were obtained from BRCA and non-BRCA. Preneoplastic cell lines were then generated by hTERT, p53 mutation (R175H), and HPVE7. To mimic the genetic changes of in vivo HGSC, oncogenes CCNE1 and PIK3CA-H1047R were then overexpressed (OE) in these immortalized cell lines. Xenografts were generated from the transformed FTE cells lines. Agilent Seahorse assays were used to study the fuel dependency, oxidative phosphorylation, and glycolysis.

Results: FTE-BRCA1 and FTE-BRCA2 cells showed differences in both glycolytic and oxidative metabolism depending on the oncogenic type. The transformed cells had lower oxidative metabolism and there was also discrepancy in the dependency of metabolites utilized by these cell lines for energy. As seen by mRNA, FTE-BRCA cells had more PCK2 protein and were characterized by higher metabolic rates; these observations were augmented in the presence of PIK3CA-H1047R. However, these FTE preneoplastic and oncogene transformed cells showed no difference in their mitochondrial mass or membrane potential.

Conclusion: FTE-BRCA mutant cells utilize glycolysis or gluconeogenesis pathways to overcome metabolic stress depending on which oncogene is driving carcinogenesis. Further work is required to elucidate the mechanism of PCK2 and BRCA link in ovarian cancer carcinogenesis.

Abstract B12: Differences in neoplastic transformation potential between OSE and FTE

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Introduction: The most common and aggressive histotype of ovarian cancer is high-grade serous carcinoma (HGSC), which accounts for approximately 90% of deaths. Furthermore, the risk of developing HGSC is increased by having a mutation in the BRCA1 and BRCA2 gene. There is considerable evidence detailing the fallopian tube epithelia (FTE) as the site of origin of the disease, yet the ovarian surface epithelium (OSE) is still proposed as the site of origin by some. Currently, no studies have compared in vivo/xenografts models derived from FTE and OSE of BRCA mutation carriers. We suggest that in vivo/xenograft models can prove a useful tool for understanding the disease origins, meanwhile accounting for the genetic characteristics of the disease.

Methodology: Immortalized and transformed cell lines, from both FTE and OSE, were derived from both BRCA1 and BRCA2 mutation carriers and non-carriers. In particular, 16 OSE cell lines were created–3 from BRCA1 carriers and noncarriers. Cell lines were transfected with hTERT and SV40, and either vectors overexpressing cMYC and hRASV12. 8 of these lines with cMYC or hRASV12 were injected into the mammary fat pad of 6-week-old female NSG mice (NOD.Cg-Prkdcscid). Similarly, 16 FTE lines were generated from 2-BRCA1 and 1 BRCA2 heterozygous mutation and 3 non-BRCA carriers overexpressing hTERT and SV40 with either cMYC or hRASV12.

Results: To date, tumors have developed from mice injected with ovarian surface epithelial cells and fallopian tube epithelial cells at the mammary fat pad and the intraperitoneal cavity. FTE cells injected intraperitoneally produced ascites and displayed tumors lining the abdominal cavity and major organs. The parental immortal lines of 4 cases were karyotyped: FTE-37 and FTE-19 (controls); FTE-13 (BRCA2 mutant) and FTE-98 (BRCA1 mutant) cell lines demonstrated varying types of tetraploidy while the control (BRCA wild type) had fewer tetraploid chromosomes. Western blot analyses demonstrated that tumors express PAX8 and epithelial markers associated with HGSC. STR profiling using GenePrint 10 also revealed that injected FTE cells grown in vitro clustered with xenograft tumors of the same cell line, demonstrating that cell line and tumors retained the same characteristics during tumor development. It is hypothesized that SV40 alone is sufficient to stimulate genomic instability in the FTE cells. Several cell lines grown in culture with expression of hRASV12 demonstrated increased proliferation relative to normal control cells.

Abstract B098: Intra-Caribbean Island differences drive breast cancer outcomes in US Caribbean-immigrant women compared to US-born Black women

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Introduction The Caribbean accounts for half of all foreign-born blacks in the USA. The biggest contributors to this immigrant population come from Cuba, Dominican Republic (DR), Jamaica, Haiti and Trinidad and Tobago. Breast cancer (BC) is the most commonly diagnosed cancer in US Black women and the second cancer related cause of death. Breast cancer in the most common cause of death of native Caribbean women. In our previous work, we found that Caribbean immigrants with breast had a better overall survival than US born Blacks. The primary objective of this study was to investigate the etiology and outcomes within the most populace Caribbean born immigrants compared to US born Black women within our cohort. Methods Data were obtained from Jackson Memorial Health Systems and University of Miami Health System Tumor Registry. Data from 1,082 Black patients were used to estimate hazard rations (HRs) of women born in Haiti, Jamaica, Bahamas, Cuba, Dominican Republic or the US born (USB) using Cox proportional hazards regression analysis for overall survival. Clinicopathologic and treatment characteristics of women from each island were compared to USB using Chi Squared testing and independent sample t-test. Results The sub-cohort contained data from Haitian (250), Jamaican (89), Bahamian (43), Dominican (38), Cuban (38) and 624 USB women. Compared to USB (57.6 years), of all the sub-groups the Bahamians were the youngest (50.5 years, P <0.001) and presented at the highest advanced stage in the cohort (54.3% vs USB 35.3%; P=0.02). Cubans (26.5%), Jamaicans (26.2%), and Haitians (25.8%) had a higher prevalence of HER2+ breast cancer in contrast to USB (17.7%), Bahamians (18.9%) and Dominicans (18.9%). Surgery was a favorable factor for survival in USB (aHR=0.26 (0.19-0.36), p<0.001), Bahamians (aHR=0.05 (0.01-0.47), p=0.008) and Jamaicans (aHR=0.08 (0.03-0.24), p<0.001). Haitian were the only immigrant group with a significant favorable outcome from radiation therapy (aHR=0.45, 95% 0.27-0.77; P=0.004). Cubans and Dominicans had a significant favorable response to surgery (aHR=0.25 (0.04-1.58), p=0.14) and radiation (aHR=0.48 (0.05-4.19), p=0.51) but did not reach statistical significance due to small sample size. Conclusion This data demonstrates and highlights intra-island and ethnic differences linked to clinicopathologic features of breast cancer in Black women living in the US. Further studies are needed to identify biological factors impacting the etiology and outcomes breast cancer in the immigrant Caribbean population and US born Black.

Abstract B086: Short-term effects of immigration on the development of breast cancer in women of African descent

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Introduction Haitians diagnosed with breast cancer in Haiti experience a significantly worse outcome than immigrants in Miami that appears to be related to 1) more advanced stage, 2) younger age, 3) more ER-negative tumors, and 4) lack of timely effective treatments including restricted access to trastuzumab and radiation. The objective of the study was to evaluate the epigenetic differences by DNA methylation between women of African descent who have breast cancer and are native to Haiti, the USA or who have emigrated from Haiti to the USA. Methods The study was IRB approved and conducted between 2017-2019 at University of Miami (n=50), Ministry of Health Haiti and Innovating Health International (n=35) and Moffit Cancer Center (n=14). DNA were extracted from saliva (DNAGenotek PrepIT-LP), FFPE breast tumor and normal adjacent breast epithelia (QIAmp DNA FFEP kit) when available. Reduced representation bisulfite sequencing was performed by BGI Inc (Cambridge MA) with EZ DNA metylation gold kit (Zymo) and followed by directional BS-sequence library preparation on HISeq x Ten, PE150. Data was processed in methylKit package and analyzed in a two-step filtering process to 1) include 200bp tiled regions to maximize CpG coverage, > 10 read coverage, <400 read coverage, >3 shared CpGs per group or all samples if the number of samples was less than 3 in the group, q <0.05 and 2) study DMRs only in ER+ breast tumors. Results The mean age of Haitian natives (H) (49.4 years), Haitian Immigrants (HI) (51.9 years) and African American (AA) (48.6 years). The H were in the US for a range of 5-47 years. 64.7% of H, 63% of HI and 42.9% of AA were diagnosed at stages III/IV. Differentially methylated regions (DMRs) were assessed between groups for germline, tumor and normal breast epithelial. There were significantly more DMRs in germline DNA than tumor for each comparison group. The DMRs identified in germline DNA; AA vs HI=54,150 DMRs, HI vs H=45,031 and H vs AA=40,461 DMRs. In the tumors, there were more DMRs between HI and AA (556 DMRs) than H vs AA (33 DMRs) and H vs AA (3 DMRs). Pathway analysis of DMRs identified in germline DNA amongst the three groups revealed common tumor molecular mechanisms of cancer, AMPK and CREB signaling. Whereas Wnt3a, EZH2, PI3K and molecules involved in DNA damage and cell cycle were distinct effectors in tumors of each group. Conclusion This work provides data for exploring the biological basis of breast cancer subtypes within the US Black sub-populations stratified by time and nativity. Haitian immigrants and US born Black women with breast cancer have more epigenetic differences and represent unique pathways which drive tumor biology.

Abstract A82: Ovarian cancer cells hijack immune functions of omental milky spots for metastatic colonization

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Introduction: The omentum is the primary site of metastasis in both ovarian cancer models and clinical disease. It is composed predominantly of adipose studded with lymphoreticular organs (milky spots), distinguishing it from other peritoneal adipose. Milky spots are specialized for immune cell trafficking and peritoneal surveillance. We and others have shown that ovarian cancer cells exploit the physiologic function(s) of these structures for omental metastatic colonization. The purpose of this study was to identify cellular and molecular mechanisms responsible for ovarian cancer homing to and growth within milky spot structures.

Experimental Procedures: Quantitative in vivo and ex vivo assays were used to evaluate human (SKOV3ip.1, HEYA8, and CaOV3) and murine (ID8) ovarian cancer cell localization to milky spots on the murine omental fat band. Assays were conducted using C57/Bl6 mice or those lacking B cells (Igh6^-/-); T cells (Nude); B and T cells (Rag1^-/-); or B, T, and NK cells (BN XID). In vitro assays were used to assess the migration-promoting activity of omentum- and macrophage-conditioned media. Standard approaches were used to assess protein expression, cell growth and viability, etc.

Rationale: Milky spots provide resident tissue macrophages and lymphocytes needed for peritoneal homeostasis. Macrophages and stromal cells secrete chemokines promoting peritoneal lymphocyte homing to the omentum. In response to peritoneal irritants, activated CD11b⁺ milky spot macrophages organize coordinated expansion of vascular and stromal compartments. The increase in both the number and size of milky spots is needed to process particulates, resolve infections, and encapsulate foreign bodies.

Hypothesis: CD11b+ cells secrete homeostatic chemokines promoting G-protein-dependent migration of ovarian cancer cells to milky spots. Ovarian cancer cell binding to adhesion molecules on the milky spot surface activates CD11b+ cell-dependent tissue remodeling, creating a microenvironment promoting ovarian cancer growth.

New Findings: Consistent with our hypothesis, in vivo assays showed that macrophage depletion prior to injection of ID8 and SKOV3ip.1 cells prevented microscopic metastasis formation. In vitro assays found that macrophages are required for ovarian cancer cell localization to milky spots. These data prompt the hypothesis that CD11b⁺ cells produce a factor(s) responsible for the migration-promoting ability of omentum-conditioned media.

To test this, media was conditioned by omental adipose isolated from mice after macrophage depletion. In support of our hypothesis, the migration-promoting activity of macrophage-depleted omentum-conditioned media was on a par with that of media conditioned by milky spot-deficient adipose. Further, media conditioned by CD11b⁺ cells recapitulates the migration-promoting activity exhibited by omentum-conditioned media.

In vitro and ex vivo assays were used to test whether ovarian cancer cells utilize mechanisms analogous to lymphocyte trafficking for milky spot homing. Specifically, cells were pretreated with pertussis toxin or vehicle alone, and then evaluated for migration in response to omentum-conditioned media and milky spot localization ex vivo. Consistent with data reported for lymphocyte homing, pertussis toxin pre-treatment caused a 40% to 50% reduction in ovarian cancer cell homing.

Conclusions and Current Efforts: Our data support a model in which CD11b+ macrophages secrete one or more chemokines promoting G-protein receptor-dependent ovarian cancer cell migration, and potentially integrin activation, which mediate milky spot homing. Current experiments focus on identification of integrin-ligand interactions, CD11b+ cell activation, and defining the link between ovarian cancer cell growth and increase in milky spot size and number.

Abstract A63: Estrogen receptor signaling in fallopian tube epithelia of BRCA mutation carriers

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A survey of BRCA1, BRCA2, and PALB2 mutations in women with breast cancer in Trinidad and Tobago

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The mortality rate from breast cancer in the nation of Trinidad and Tobago is among the highest of any country in the Caribbean region. The contribution of inherited gene mutations to the burden of breast cancer in Trinidad and Tobago has not been studied. We examined the prevalence of mutations in three susceptibility genes (BRCA1, BRCA2, and PALB2) in breast cancer patients in Trinidad and Tobago. We studied 268 unselected breast cancer patients from Trinidad and Tobago and looked for mutations across the entire coding sequences of BRCA1, BRCA2, and PALB2. Overall, 28 of 268 patients (10.4 %) had a mutation in one of the three genes, including 15 in BRCA1, ten in BRCA2, two in PALB2, and one in both BRCA2 and PALB2. There were 25 different mutations identified; of these, four mutations were seen in two patients each. Given the high prevalence of mutations, it is reasonable to offer genetic testing for these three genes to all breast cancer patients in Trinidad and Tobago.

A high frequency of PALB2 mutations in Jamaican patients with breast cancer

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Purpose: Jamaica is an island nation with one of the highest breast cancer incidence rates in the Caribbean (40/100,000 per year). The contribution of cancer susceptibility gene mutations to the burden of breast cancer in Jamaica has not yet been explored. We sought to determine the prevalence of germline mutations in BRCA1, BRCA2, and PALB2 in 179 unselected Jamaican women with breast cancer.

Methods: We sequenced the entire coding regions of BRCA1, BRCA2, and PALB2 for all the study subjects.

Results: Overall, 8 of 179 patients (4.5%) had a mutation in one of the three genes: one in BRCA1, two in BRCA2, and five in PALB2.

Conclusions: These data suggest that in addition to BRCA1 and BRCA2, PALB2 should be included in genetic testing for breast cancer patients in Jamaica.

Walking the Maternal Tightrope: Work and Family in America

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In the last few decades, an unprecedented number of women with children have entered the US workforce. The ability to negotiate the roles of parent and employee is important to the health and financial well-being of these women and their families, but institutional and social barriers impede the process. Using the empirical and theoretical literature on women and work, this article examines these barriers. The authors address the impact of cultural ideals, psychological processes, and public policy on the maternal work-family balance. Several changes that would help create an atmosphere supportive of balance are explored, including increased support for shared parenting and improvements to the Family and Medical Leave Act.

Sacrifices, Sisterhood, and Success in the Ivory Tower

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As institutional, Black women and faculty, structural we are barriers acutely to aware the career of the success numerous of Black individual, women institutional, and structural barriers to the career success of Black women in higher education. This awareness, along with a general lack of resources and support, prompted us to form WellAcademic-a company focused on helping faculty, administrators, and graduate students, particularly underrepresented minorities, increase their productivity and well-being. In addition to individual coaching and workshop facilitation on publishing, stress management, and dissertation completion, we are committed to providing low-to no-cost resources via our blog, articles, and, monthly newsletter. So, we are excited to have the opportunity to publish this piece together and share with other Black women faculty strategies we have learned that may help others thrive in the academy.