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Saracatinib inhibits proliferation and c-Myc, and GLUT1 in MFPs of ER- MMTV-neu* mouse model.

Preventing breast cancer will require the development of targeted strategies that can effectively block disease progression. Tamoxifen and aromatase inhibitors are effective in addressing estrogen receptor–positive (ER+) breast cancer development, but estrogen receptor–negative (ER−) breast cancer remains an unmet challenge due to gaps in pathobiologic understanding. In this study, we used reverse-phase protein array to identify activation of Src kinase as an early signaling alteration in premalignant breast lesions of women who did not respond to tamoxifen, a widely used ER antagonist for hormonal therapy of breast cancer. Src kinase blockade with the small-molecule inhibitor saracatinib prevented the disorganized three-dimensional growth of ER− mammary epithelial cells in vitro and delayed the development of premalignant lesions and tumors in vivo in mouse models developing HER2+ and ER− mammary tumors, extending tumor-free and overall survival. Mechanistic investigations revealed that Src blockade reduced glucose metabolism as a result of an inhibition in ERK1/2–MNK1–eIF4E–mediated cap-dependent translation of c-Myc and transcription of the glucose transporter GLUT1, thereby limiting energy available for cell growth. Taken together, our results provide a sound rationale to target Src pathways in premalignant breast lesions to limit the development of breast cancers. Cancer Res; 75(22); 4863–75. ©2015 AACR.

RPPA data showing fold changes in transcriptional regulator expressions in saracatinib treated 10A.B2 cells over vehicle control.

Description of additional methods and procedures used in the study. Also includes Supplementary References.

Legend for Supplementary Table S1 and Supplementary Figures S1-S5.

Simple Summary Genomic imprinting is an inherited form of parent-of-origin specific epigenetic gene regulation that is dysregulated by poor prenatal nutrition and environmental toxins. Here, we showed that KCNK9 is imprinted in breast tissue and identified the differentially methylated region (DMR) controlling its imprint status. Hypomethylation at the DMR, coupled with biallelic expression of KCNK9, occurred in 63% of triple-negative breast cancers (TNBC). The association between hypomethylation and TNBC status was highly significant in African-Americans (p = 0.006), but not in Caucasians (p = 0.70). The high frequency of KCNK9 DMR hypomethylation in TNBC and non-cancerous breast tissue from high-risk women provides evidence that hypomethylation of the KNCK9 DMR/TASK3 overexpression may provide a new target for prevention of TNBC. Abstract Genomic imprinting is an inherited form of parent-of-origin specific epigenetic gene regulation that is dysregulated by poor prenatal nutrition and environmental toxins. KCNK9 encodes for TASK3, a pH-regulated potassium channel membrane protein that is overexpressed in 40% of breast cancer. However, KCNK9 gene amplification accounts for increased expression in

PURPOSE: Here we aim to review the association between mammographic density, collagen structure and breast cancer risk. FINDINGS: While mammographic density is a strong predictor of breast cancer risk in populations, studies by Boyd show that mammographic density does not predict breast cancer risk in individuals. Mammographic density is affected by age, parity, menopausal status, race/ethnicity, and body mass index (BMI). New studies normalize mammographic density to BMI may provide a more accurate way to compare mammographic density in women of diverse race and ethnicity. Preclinical and tissue-based studies have investigated the role collagen composition and structure in predicting breast cancer risk. There is emerging evidence that collagen structure may activate signaling pathways associated with aggressive breast cancer biology. SUMMARY: Measurement of film mammographic density does not adequately capture the complex signaling that occurs in women with at-risk collagen. New ways to measure at-risk collagen potentially can provide a more accurate view of risk.

Obesity is a highly prevalent and modifiable breast cancer risk factor. While the role of obesity in fueling breast cancer progression is well established, the mechanisms linking obesity to breast cancer initiation are poorly understood. A hallmark of breast cancer initiation is the disruption of apical polarity in mammary glands. Here we show that mice with diet-induced obesity display mislocalization of Par3, a regulator of cellular junctional complexes defining mammary epithelial polarity. We found that epithelial polarity loss also occurs in a 3D coculture system that combines acini with human mammary adipose tissue, and establish that a paracrine effect of the tissue adipokine leptin causes loss of polarity by overactivation of the PI3K/Akt pathway. Leptin sensitizes non-neoplastic cells to proliferative stimuli, causes mitotic spindle misalignment, and expands the pool of cells with stem/progenitor characteristics, which are early steps for cancer initiation. We also found that normal breast tissue samples with high leptin/adiponectin transcript ratio characteristic of obesity have an altered distribution of apical polarity markers. This effect is associated with increased epithelial cell layers. Our results provide a molecular basis for early alterations in epithelial architecture during obesity-mediated cancer initiation. ispartof: ONCOGENE vol:38 issue:20 pages:3855-3870 ispartof: location:England status: published

Black/African-American (AA) women, relative to their White/European-American (EA) counterparts, experience disproportionately high breast cancer mortality. Central to this survival disparity, Black/AA women have an unequal burden of aggressive breast cancer subtypes, such as triple-negative breast cancer (ER/PR-, HER2-wild type; TNBC). While TNBC has been well characterized, recent studies have identified a highly aggressive androgen receptor (AR)-negative subtype of TNBC, quadruple-negative breast cancer (ER/PR-, HER2-wildtype, AR-; QNBC). Similar to TNBC, QNBC disproportionately impacts Black/AA women and likely plays an important role in the breast cancer survival disparities experienced by Black/AA women. Here, we discuss the racial disparities of QNBC and molecular signaling pathways that may contribute to the aggressive biology of QNBC in Black/AA women. Our immediate goal is to spotlight potential prevention and therapeutic targets for Black/AA QNBC; ultimately our goal is to provide greater insight into reducing the breast cancer survival burden experienced by Black/AA women.

Over 90% of breast cancer is cured; yet there remain highly aggressive breast cancers that develop rapidly and are extremely difficult to treat, much less prevent. Breast cancers that rapidly develop between breast image screening are called "interval cancers." The efforts of our team focus on identifying multiscale integrated strategies to identify biologically aggressive precancerous breast lesions. Our goal is to identify spatiotemporal changes that occur prior to development of interval breast cancers. To accomplish this requires integration of new technology. Our team has the ability to perform single cell in situ transcriptional profiling, noncontrast biological imaging, mathematical analysis, and nanoscale evaluation of receptor organization and signaling. These technological innovations allow us to start to identify multidimensional spatial and temporal relationships that drive the transition from biologically aggressive precancer to biologically aggressive interval breast cancer. This article is categorized under: Cancer > Computational Models Cancer > Molecular and Cellular Physiology Cancer > Genetics/Genomics/Epigenetics.

During puberty, a woman’s breasts are vulnerable to environmental damage (“window of vulnerability”). Early exposure to environmental carcinogens, endocrine disruptors, and unhealthy foods (refined sugar, processed fats, food additives) are hypothesized to promote molecular damage that increases breast cancer risk. However, prospective human studies are difficult to perform and effective interventions to prevent these early exposures are lacking. It is difficult to prevent environmental exposures during puberty. Specifically, young women are repeatedly exposed to media messaging that promotes unhealthy foods. Young women living in disadvantaged neighborhoods experience additional challenges including a lack of access to healthy food and exposure to contaminated air, water, and soil. The purpose of this review is to gather information on potential exposures during puberty. In future directions, this information will be used to help elementary/middle-school girls to identify and quantitate environmental exposures and develop cost-effective strategies to reduce exposures.

There is a critical need for additional and effective comprehensive programs to increase the number of underrepresented minority students pursuing scientific careers. Science education for high school students is often fragmented and delivered with single-focused approaches, for example traditional classroom lectures, hands-on-activities, or conducting research. The current paper examines a comprehensive biomedical research program that integrated classroom teaching, hands-on-activities, conducting a research study, and mentoring from scientists in authentic scientific settings. The expectation is that a comprehensive program would offer students a well-rounded experience and skills in science. We assessed short-term psychosocial outcomes and long-term academic outcomes in the participants, largely minority high school students, who are traditionally underrepresented in science. The psychosocial outcomes assessed pre- and post-program include: knowledge of science pathways, self-efficacy in science, attitudes toward science, and scientific communication skills. Although non-significant, post-program results showed an increasing trend for knowledge of science pathways, self-efficacy in science, and attitudes toward science. Post-program, students reported statistically significant increases in feeling they had role models in science. A long-term assessment of 31 participating students showed that 77% of these students were attending college, 79% were majoring in STEM, and 75% were planning to pursue additional higher education. Our findings provide evidence for the short-term and long-term benefits of a comprehensive biomedical research program conducted in an authentic scientific setting.

Despite cancer being the leading cause of death across most racial/ethnic groups, Hispanic women have the second highest mortality rate attributed to diabetes (4.7%) according to the Centers for Disease Control and Prevention (CDC). While cancer and diabetes are two distinct diseases, previous studies have demonstrated that diabetic women have a poor chance of breast cancer survival when compared to nondiabetic women. Well-known key drivers of hyperinsulinemia and insulin resistance, such as insulin and AMPK, are also those involved in breast cancer. This link could possibly contribute to the increased mitogenic effects and risk for aggressive breast cancers in Hispanic women. Based on these findings, metformin, a drug standardly used to treat and prevent hyperglycemia, may be a possible alternative (other than tamoxifen) for breast cancer prevention. Eat, Move, Live (EML), a 5-week community-based program, focuses on targeting possible treatments of chronic diseases and risk reduction through attitude and lifestyle modifications. Exercise, nutritional and health awareness classes were implemented to change participants' perspectives regarding chronic diseases and their susceptibility to other morbidities. Questionnaires were given to the participants at baseline and at two follow-ups (5 weeks and 12 weeks) to assess any changes in their attitudes, behaviors, nutrition, lifestyle and beliefs around taking medication for preventative treatments. A total of 56 participants' pretreatment responses were collected via a five-point Likert scale (1-strongly disagree, 5-strongly agree). Demographic data showed that 69% of the respondents were Hispanic women, of whom 46% completed an education level of high school or less. A majority of the responses averaged a “neutral” response to taking medication for management and prevention of diabetes. We infer that their inability to select a stance in their responses may be associated with the lack of knowledge that the community has regarding chronic diseases and risk-prevention methods. Therefore, we anticipate that availability of proper education tools and resources is essential to potentially prevent future morbidities and mortalities. Ultimately, we aim to establish a pilot study that emphasizes the necessity and importance of interventional programs, like EML, to enhance chemoprevention using metformin and improve health outcomes in high-risk breast cancer populations. Citation Format: Mayra Serrano, Angelica Sanchez, Christine Thai, Katty Nerio, Cristal Resto, Marisela Garcia, Tanya A. Chavez, Laura L. Kruper, Veronica C. Jones, Lisa D. Yee, Alan Nuñez, Ellen J. Rippberger, Angela K. Wong, Noé R. Chávez, Karen Herold, Chidimma M.K. Kalu, Jackelyn A. Alva-Ornelas, Jerneja Tomsic, Krista M. Round, Regina Agulto, Margarita Robles, Ombeni M. Idassi, Kendall J. Kennedy, Christopher Sistrunk, Victoria L. Seewaldt. Receptiveness of metformin as a breast cancer prevention drug within the Hispanic community [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr B119.

The United States is increasingly racially and ethnically diverse. In fact, California is now a majority-minority region, with a greater percentage of its population comprised of racial minorities than whites. Yet, minorities are continually under-represented in clinical research trials, which provide crucial information on which the future of cancer treatments is built. Without representative inclusion of participants of color in clinical research, we cannot develop effective preventative and treatment approaches for everyone. This current study investigates the factors, including characteristics of study consenters, that may influence women—particularly women of color (WOC)—to accept or decline participation in breast cancer-related trials. We assess these factors through a brief survey, administered to patients immediately after they were invited to participate in a breast cancer-related clinical study. From the beginning of study accrual to the present, twenty-three patients have taken the survey. We anticipate accruing 200 participants at a rate of 25 per month. For the preliminary analyses, we split participants in two groups: white women (WW) (n = 14) and women of color (WOC) (n = 9). We conducted independent sample t-tests to compare the responses of WW and WOC. More WOC (M = 1.44, SD = 0.73) reported that it is important that their consenter is of the same ethnicity or race than WW (M = 1.00, SD = .00), t (21) = -2.32, p < .05. Similarly, WOC (M = 1.44, SD = .73) also reported that it is important that the person inviting them to participate in research look like people in their community, compared to the importance placed on this factor by WW (M = 1.00, SD = .00), t (21) = -2.32, p < .05). More WOC (M = 2.33, SD = 1.23) also cited “feeling overwhelmed” with their medical condition as influential in their decision to participate in clinical research than WW (M = 1.31, SD = .48), t (20) = -2.75, p < .05. Although both groups positively rated their interaction with the consenter, we observed marginal differences between WOC and WW. WOC (M = 7.00, SD = .00) gave higher ratings to the variable of “consenter created an atmosphere of trust and support” compared to ratings given by WW (M = 6.29, SD = 1.07), t (21) = -1.99, p = .06. Though participants are generally satisfied with their consenter interaction, different factors influence WW and WOC as they decide whether to participate in clinical research. When identified, these factors can be used to inform more inclusive consenting processes. Citation Format: Noe R. Chavez, Alan Nunez, Angela K. Wong, Tanya A. Chavez, Ellen Rippberger, Christine Thai, Angelica Sanchez, Ombeni M. Idassi, Krista M. Round, Kendall Kennedy, Margarita Robles, Jackelyn A. Alva-Ornelas, Jerneja Tomsic, Chidimma M.K. Kalu, Laura L. Kruper, Veronica C. Jones, Sharon Clancy, Amy C. Polverini, Courtney Vito, Karen Harold, Terry Hyslop, Carola M. Zalles, Daniel B. Schmolze, Christopher Sistrunk, Victoria L. Seewaldt. Influencing women's attitudes toward participation in breast cancer clinical research: Improving inclusion of women of color [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr A082.

The United States (US) is racially and culturally diverse. Still, white women disproportionately comprise clinical trial participants, stemming largely from the fact that minorities often decline participation. Clinical trial findings, therefore, have not been representative of the diverse patient population, and have stymied progress in precision medicine and cancer prevention. According to the CDC, among Asian/Pacific Islander females, the leading cause of death is cancer. Moreover, they have more total cancer-related deaths than any other racial group. Per the 2017 US Census, Asians are the third largest minority group of the population (6.6%). Despite the grave health disparities faced by a sizable proportion of the population, the literature is sparse with regards to their research engagement. For example, several studies have focused on Black and Hispanic consenting rates, including one study that demonstrated no significant differences between the two groups (Wendler, Kington, Madans, Wye, Christ-Schmidt, Pratt et al., 2005) and another that indicated relatively lower enrollment rates among Hispanics and Blacks (Murthy, Krumholz, Gross, 2004). This gap in research is largely due to the marked heterogeneity with respect to language, nationality, and acculturation status. We examined Asian participation in three therapeutic, noninterventional trials that aim to develop early detection methods and accurate prognosis in women at high risk for breast cancer. Primarily, we collect leftover tissue and/or cells from fine needle aspirations (FNA) to better understand the cellular microenvironment that correlates with breast cancer development. We analyzed demographic data across these studies to investigate refusal rates among Asian women. Across the three trials, 3,119 participants were screened. Of the total number screened, 426 participants were eligible, 368 consented, and 58 declined. Of those who declined, 23.5% were Asians, 13.9% Hispanic, 8.3% Blacks, 5.6% Native Americans, and 11.4% White/non-Hispanic. Notably, Asians had the highest refusal rate. Fisher's exact test (two-tailed) was conducted to examine refusal rates for Asians compared to Whites, Blacks, and Hispanics. Results suggest that Asians had significantly higher rates of compared to Whites (p = .02, observed odds ratio = .42, 95% CI [.20, .86]), but were not significantly different than Blacks or Hispanics. The under-representation of Asians in research may exacerbate health disparities. Thus, further studies should examine ways, such as increasing racial and cultural competency, for increasing Asian representation in clinical research studies. Ultimately, we believe that illuminating under-representation of the “forgotten” Asian population in clinical research can inform future interventions that promote chemoprevention and treatment of this high-risk population. Citation Format: Stacey N. Doan, Christine Thai, Angela K. Wong, Tanya A. Chavez, Angelica Sanchez, Laura L. Kruper, Veronica C. Jones, Sharon Clancy, Amy C. Polverini, Lisa D. Yee, Courtney Vito, Alan Nunez, Ellen J. Rippberger, Noe R. Chavez, Karen Herold, Chidimma M.K. Kalu, Jackelyn A. Alva-Ornelas, Jerneja Tomsic, Krista M. Round, Margarita Robles, Ombeni M. Idassi, Kendall J. Kennedy, Christopher Sistrunk, Victoria L. Seewaldt. The forgotten race: Under-representation of Asians in clinical research [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr A084.

Purpose: To highlight the importance of building a high-risk breast cancer clinic for women who do not have a BRCA 1, BRCA 2 or other highly penetrant cancer susceptibility mutation. Background: Breast cancer is the most common cancer in women and the second most prevalent cause of cancer death of women in the United States; the lifetime risk for breast cancer in women is approximately 12%. Women may be at increased risk for breast cancer for many reasons including family history, genetic alterations, age, reproductive status and menstrual history. Most women who are at increased risk of developing breast cancer do not have a BRCA 1, BRCA 2 or other mutation. The majority of breast cancer diagnoses are due to acquired somatic mutations; only 5 to 10% of breast cancer diagnoses are attributable to highly penetrant Mendelian cancer susceptibility genes. White women with Ashkenazi Jewish ancestry tend to have a higher incidence of BRCA 1 and 2 mutations; traditionally, most research efforts about highly penetrant genes have been focused on this group rather than other racial and ethnic groups. However, there is a great need to study breast cancer risk-reduction strategies in racial and ethnic minorities in the United States, particularly because most breast cancers are not caused by BRCA 1 and 2 mutations. City of Hope is located approximately 21 miles northeast of Los Angeles and operates 13 clinical practice locations including Los Angeles, Orange, Riverside, San Bernardino and Ventura counties. These five counties are home to the majority of California's multicultural and ethnic residents where San Bernardino County has the highest percentage of Hispanics (49.9%) and blacks (8.3%), Ventura County has the highest percentage of whites (48.1%), and Orange County has the highest concentration of Asians (18.2%). It has been established in the literature that the greatest benefit from breast cancer prevention strategies comes from treating women who are at high risk of the disease. While it is important to build a high-risk breast cancer clinic for women with genetic mutations, it is equally important to build a high-risk breast clinic for women who are at increased risk of breast cancer but do not have a mutation, particularly because most breast cancer is diagnosed in this population. In addition, it is crucial to educate high-risk patients that although they may have tested negative for a genetic mutation if they have a family history of breast cancer, they warrant close clinical surveillance. Methods: We are proposing a retrospective, descriptive study of data that will be collected as part of a high-risk breast cancer program implemented by City of Hope. Results/Conclusions: We expect to discuss the findings related to serving women of all races and ethnicities who do not have a mutation in a highly penetrant gene mutation. Citation Format: Karen Herold, Lisa D. Yee, Chidimma M. Kalu, Laura L. Kruper, Veronica C. Jones, Amy C. Polverini, Sharon Clancy, Tanya A. Chavez, Jackelyn A. Alva-Ornelas, Noe R Chavez, Ellen J. Rippberger, Jerneja Tomsic, Christopher Sistrunk, Ombeni Idassi, Daniel B. Schmolze, Courtney Vito, Alan Nunez, Angela K. Wong, Krista M. Round, Christine Thai, Angelica Sanchez, Margarita Robles, Kendall Kennedy, Terry Hyslop, Victoria L. Seewaldt. Architecture of increased breast cancer risk [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr B109.

Background: Breast cancer (BC) prevention clinical trials (CTs) play a vital role in the progress of preventative measures and treatments for all races and ethnicities. However, Northern European whites (NE/W) continue to be disproportionally enrolled (e.g., 93.5% were non-Hispanic white in the STAR trial), while minorities such as Asians, blacks, Latinas, and Native Americans (NA) lag in participation. Current studies suggest that minorities are not approached as frequently as NE/W; however, they are just as willing to participate. Here we present a successful recruitment strategy to improving minority accrual in CTs at a Comprehensive Cancer Center located in Duarte, CA. Method: Results from community focus groups suggested the need to mentor local youth who strive to pursue a career in the medical field. Consequently, from February 2016 to July 2018, four bilingual, bicultural clinical research assistants (CRAs) were recruited from the catchment area of City of Hope (CoH). The CRAs, in collaboration with seven surgeons, two radiologists, and one medical oncologist, led the recruitment for three nontherapeutic BC prevention CTs at CoH. Results: All four CRAs were 1) first-generation American, 2) fluent in Spanish or Vietnamese, 3) born and raised in Southern California, and 4) pre-health. Of the 3,148 patients who were screened, 398 were eligible for enrollment, 369 consented, and 58 declined. Primary languages and races/ethnicities of those who declined include the following: 7% Armenian, 9% Chinese, 78% English, 2% Thai, and 5% Spanish; 28% Asian, 3% black, 28% Latina, 2% NA, and 67% white (22% NE, 17% Middle Eastern/North African). Demographics of the consenting population include the following: primary language - >1% Armenian, 4% Chinese, 89% English, >1% Korean, and 7% Spanish; race/ethnicity - 14% Asian, 6% black, 30% Latina, 5% NA, and 75% white (40% NE). Of the white population (n = 277), 11% were Middle Eastern/North African, 53% NE, and 36% Latina. Accrual surpassed both the CoH catchment area (11.3% Asian, 8% black, 24% Hispanic, 1% NA, and 32% NE/W) and the CoH interventional/nontherapeutic CT population (10% Asian/Pacific Islander, 4% black, 21% Hispanic, >1% NA, and 55% NE/W). Conclusion: Contrary to current accrual of CTs, here we show that minorities can have a large representation in CT accrual, as long as they are provided the opportunity. Accrual of Asians, Latinas, and NAs exceeded the catchment area and accrual of other CoH CTs. Interestingly, Chinese-speaking women comprised the highest declination group of the non-English speakers, and Asians and Latinas declined the most outside of non-whites. Cultural competency and bilingualism appear to be characteristics of a CRA that may help in accruing minority women into CTs. Our findings suggest that they are just as willing to participate, and the first step is to simply ask. Citation Format: Tanya A. Chavez, Christine Thai, Angelica Sanchez, Laura L. Kruper, Veronica C. Jones, Sharon Clancy, Amy C. Polverini, Lisa D. Yee, Courtney A. Vito, Noé R. Chávez, Alan Nuñez, Ellen J. Rippberger, Angela K. Wong, Karen Herold, Chidimma M.K. Kalu, Jackelyn A. Alva-Ornelas, Jerneja Tomsic, Krista M. Round, Margarita Robles, Ombeni Idassi, Kendall J. Kennedy, Terry Hyslop, Carola M. Zalles, Christopher Sistrunk, Victoria L. Seewaldt. Diversifying breast cancer clinical trial accrual: An approach to recruitment at a Comprehensive Cancer Center [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr A083.

A subset of cells, termed side-population (SP), which have the ability to efflux Hoeschst 33342, have previously been demonstrated to act as a potential method to isolate stem cells. Numerous stem/progenitor cells have been localized in different regions of the mouse hair follicle (HF). The present study identified a SP in the mouse HF expressing the ABCG2 transporter and MTS24 surface marker. These cells are restricted to the upper isthmus of the HF and have previously been described as progenitor cells. Consistent with their SP characteristic, they demonstrated elevated expression of ABCG2 transporter, which participates in the dye efflux. Analysis of tumor epidermal cell lines revealed a correlation between the number of SP keratinocytes and the grade of malignancy, suggesting that the SP may play a role in malignant progression. Consistent with this idea, the present study observed an increased number of cells expressing ABCG2 and MTS24 in chemically induced skin tumors and skin tumor cell lines. This SP does not express the CD34 surface marker detected in the multipotent stem cells of the bulge region of the HF, which have been defined as tumor initiation cells. The present study concluded that a SP with properties of progenitor cells is localized in the upper isthmus of the HF and is important in mouse skin tumor progression.

We have previously demonstrated that ras-mediated skin tumorigenesis depends on signaling pathways that act preferentially through cyclin D1 and D2. Interestingly, the expression of cyclin D3 inhibits skin tumor development, an observation that conflicts with the oncogenic role of D-type cyclins in the mouse epidermis. Here, we show that simultaneous up and downregulation of particular members of the D-type cyclin family is a valuable approach to reduce skin tumorigenesis. We developed the K5D3/cyclin D1(-/-) compound mouse, which overexpresses cyclin D3 but lacks expression of cyclin D1 in the skin. Similar to K5D3 transgenic mice, keratinocytes from K5D3/cyclin D1(-/-) compound mice show a significant reduction of cyclin D2 levels. Therefore, this model allows us to determine the effect of cyclin D3 expression when combined with reduced or absent expression of the remaining two members of the D-type cyclin family in mouse epidermis. Our data show that induced expression of cyclin D3 compensates for the reduced level of cyclin D1 and D2, resulting in normal keratinocyte proliferation. However, simultaneous ablation of cyclin D1 and downregulation of cyclin D2 via cyclin D3 expression resulted in a robust reduction in ras-mediated skin tumorigenesis. We conclude that modulation of the levels of particular members of the D-type cyclin family could be useful to inhibit tumor development and, in particular, ras-mediated tumorigenesis.

The proto-oncogene c-Myc encodes a transcription factor that is implicated in the regulation of cellular proliferation, differentiation, and apoptosis. Myc accelerates the rate of cell proliferation, at least in part, through its ability to down-regulate the expression of the cell cycle inhibitor p27 Kip1 . Moreover, p27 Kip1 protein levels are regulated by ubiquitin-mediated turnover, leading to destruction by the E3 ubiquitin ligase SCF Skp2 . Therefore, we hypothesize that a lack of Skp2 expression should lead to increased p27 Kip1 levels and further inhibition of Myc -mediated proliferation and tumorigenesis. Myc expression in epithelial tissues of transgenic mice (K5- Myc ) led to increased keratinocyte proliferation and the development of spontaneous tumors within the oral cavity. We generated K5- Myc –transgenic mice in an Skp2-null background. Consistent with our hypothesis, we found that Myc -mediated keratinocyte hyperproliferation was abolished by the loss of Skp2. However, Skp2 ablation did not affect Myc -driven tumorigenesis because the incidence, latency, and degree of differentiation of oral tumors were identical between K5- Myc /Skp2 +/+ and K5- Myc /Skp2 −/− mice. Altogether, these findings suggest that Skp2 and p27 Kip1 are critical for Myc -driven keratinocyte proliferation; however, Myc -mediated tumorigenesis in the oral epithelium is independent of the Skp2-p27 Kip1 axis.

Desmosplasia is a characteristic of most solid tumors and leads to fibrosis through abnormal extracellular matrix (ECM) deposition, remodeling and post translational modifications. The resulting stiff tumor stroma not only compromises vascular integrity to induce hypoxia and impede drug delivery, but also promotes aggressiveness by potentiating the activity of key growth, invasion, and survival pathways. Intriguingly, many of the pro-tumorigenic signaling pathways which are mechanically activated by ECM stiffness also promote glucose uptake and aerobic glycolysis, and an altered metabolism is a recognized hallmark of cancer. Indeed, emerging evidence suggests that metabolic alterations and an abnormal ECM may cooperatively drive cancer cell aggression and treatment resistance. Accordingly, improved methods to monitor tissue mechanics and metabolism promise to improve diagnostics and treatments to ameliorate ECM stiffening and elevated mechanosignaling may improve patient outcome. Here we discuss the interplay between ECM mechanics and metabolism in tumor biology and suggest that monitoring these processes and targeting their regulatory pathways may improve diagnostics, therapy, and the prevention of malignant transformation.

Breast cancers with a basal-like gene signature are primarily triple-negative, frequently metastatic, and carry a poor prognosis. Basal-like breast cancers are enriched for markers of breast cancer stem cells as well as markers of epithelial-mesenchymal transition (EMT). While EMT is generally thought to be important in the process of metastasis, in vivo evidence of EMT in human disease remains rare. Here we report a novel model of human triple-negative breast cancer, the DKAT cell line, which was isolated from an aggressive, treatment-resistant triple-negative breast cancer that demonstrated morphological and biochemical evidence suggestive of phenotypic plasticity in the patient. The DKAT cell line displays a basal-like phenotype in vitro when cultured in serum-free media, and undergoes phenotypic changes consistent with EMT/MET in response to serum-containing media, a unique property among the breast cancer cell lines we tested. This EMT is marked by increased expression of the transcription factor Zeb1, and Zeb1 is required for the enhanced migratory ability of DKAT cells in the mesenchymal state. DKAT cells also express progenitor-cell markers, and single DKAT cells are able to generate tumorspheres containing both epithelial and mesenchymal cell types. In vivo, as few as ten DKAT cells are capable of forming xenograft tumors which display a range of epithelial and mesenchymal phenotypes. The DKAT model provides a novel model to study the molecular mechanisms regulating phenotypic plasticity and the aggressive biology of triple-negative breast cancers.

Background: There are more than 2.9 million breast cancer survivors in America today. However African American women have a 79% five-year survival while survival rates for European American women are at 92%. While early detection increases the survivability rates of most breast cancer, the standard mammogram fails to identify the earlier stages of some of the more aggressive types of breast cancer. Short-term breast cancer risks in women are increasingly being identified by Random Periareolar Fine Needle Aspiration (RPFNA). While the reproducibility of this technique is no longer the primary concern for its usage; RPFNA's small sample-sets and the inability to accurately correlate pathological and biochemical findings within the entire breast inhibits full acceptance of this technique. The purpose of this study was to determine if the utilization RPFNA accurately sampled the entire breast tissue. Methods: In this pilot study 10 high risk African American women who were high risk for breast cancer (known or suspected BRCA1 mutation carriers) had breast tissues collect by RPFNA. These samples were laser captured and the RPFNA protein expression for wide array of biomarkers (stem cell, epithelial- mesenchymal transition (EMT), proliferation, apoptosis and senescence) were assessed utilizing reverse-phase protein microarray in mammary epithelial cells. The same 10 high risk African American women either underwent prophylactic mastectomies or had a mastectomy after cancer diagnosis. Mastectomy specimens were serial sliced into 2.5 centimeter slices and 2 to 3 cubic centimeter samples were excised from a specified section of each slice of breast tissue. On average 103 samples were collected from each breast and the same (RPFNA) biomarkers were analyzed. Results: In this exploratory study, we have determined there are no significant differences between the expression of the biomarkers we have tested in RPFNA samples and the whole breast tissue sampling. Specifically we have tested for EMT markers (Vimentin and Snail), p53 and additional markers involved in proliferation and apoptosis. Conclusions: This study shows that RPFNA is an accurate measure of mammary breast epithelial and this technique directly correlates to the measurements found throughout the entire breast tissues. RPFNA did not show any significant differences in the expression of specific biomarkers tested, suggesting that RPFNA samples could be utilized to develop more robust breast cancer risk profiles. Citation Format: Christopher Sistrunk, Adrian Ambrose, Amira Carter, Victoria Seewaldt. Pilot study: Random periareolar fine needle aspiration (RPFNA) accurately depicts biochemical phenotype displayed in breast tissues of high risk African American women. [abstract]. In: Proceedings of the Seventh AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 9-12, 2014; San Antonio, TX. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2015;24(10 Suppl):Abstract nr B52.

Background: The current trend of increased survivability of women with breast cancer has been linked to increased awareness, early detection and better treatment. In fact the American Cancer Society estimate that there are more than 2.5 million breast cancer survivors living in America today. However the trends of these overall numbers are expressed quiet differently in the African American community. African American women have a 77% five-year survival rate while survival rates for European American are at 90%. It is clear that lack of access plays a role in poor survival of African American women. Other factors are hypothesized to be aggressive biology and lack of early detection strategies for triple-negative breast cancer. Methods and Results: Phase I: We developed a community outreach partnership with the African American community in Durham, NC. One of the concerns of the community was that aggressive breast cancers “appeared to come out of nowhere” and seemed to defy the conventional model of stepwise breast cancer progression. This alliance allowed the development of a partnership to use breast Magnetic Resonance Imaging (MRI) screening to evaluate the onset of breast cancer occurring in over 300 African American high-risk women (>48 month observation, 12 cancers). The results of this study corroborated the concerns of the community, that incidence of breast cancer could indeed occur in less than 6 months in women who had previously had a normal MRI. The results suggested that these aggressive breast cancers displayed an accelerated initiation and development within the patients of the local African American community, validating community observations. Phase II: The second concern raised by our community partners was the lack of prevention agents for these aggressive breast cancers. In partnership with Durham community members, our current study investigated signaling pathways activated during the initiation of triple-negative breast cancer in African American women. For our study 10 African American women who were at highest risk for breast cancer (either known or suspected BRCA1 mutation carriers) donated mastectomy tissue. Women either underwent prophylactic mastectomy or had mastectomy after a cancer diagnosis. Donated mastectomy specimens were serial sliced into 2.5 centimeter slices and 2 to 3 cubic centimeter samples were excised from specified section of each slice of breast tissue. Breast tissues were snap frozen, formalin-fixed and OCT embedded to preserve samples for further analysis. Thus far tissue tensile forces, collagen composition, metabolic and proteomic profiles have been characterized for this cohort. In addition biomarkers for stem cell, proliferation, apoptosis and senescence have been established. Conclusion: Our Durham Community/Duke Partnership has allowed us to study a unique phenotype displayed in pre- malignant breast lesion found in rapid onset breast cancers displayed in high-risk African American women. Our study highlights the power of investigators working in collaboration and partnership with the community. Citation Format: Christopher Sistrunk, Stephaine Robertson, Adrian Ambrose, Catherine Ibarra Drendall, Victoria Seewaldt. Breast cancers come out of nowhere: A two part Durham Community/Duke partnership to identify signaling pathways during the initiation of triple-negative breast cancer in African American women. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr A46. doi:10.1158/1538-7755.DISP13-A46

Background: Only 14% of African American women with triple-negative breast cancers will be alive at one year. Early detection is greatly needed. Here we aimed to investigate whether activation of biologic pathways that predict aggressive triple-negative breast cancers are also activated in atypia in high-risk African American women. Aggressive cancers are known to consume glucose avidly and produce lactic acid (rather than fully metabolize glucose via the Tricarboxylic Acid (TCA) cycle). This shift toward lactate production, even in the presence of adequate oxygen, is termed the Warburg effect. The Warburg effect is thought to be a late event in breast cancer, however, our studies in high-risk African American women provide evidence that the Warburg effect occurs during cancer initiation. This is an important observation as glucose-signaling can be readily targeted for breast cancer prevention with minimal toxicity. Here we investigated the role of the Warburg effect in breast cancer initiation in young high-risk African American women. Methods and Results: Similar to fluorodeoxyglucose, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) is a fluorescent glucose analog that can be used to track glucose uptake and glycolysis. 2-NBDG spectroscopy provides a means to track glucose metabolism in live mammary epithelial cells from high-risk women. We used 2-NBDG spectroscopy to measure glucose uptake in ER-breast cancer and live atypical mammary epithelial cells from high-risk premenopausal women. We observe that both triple-negative breast cancer and a subset of atypia exhibits accumulation of 2-NBDG. There is growing recognition that phosphoprotein signaling networks (rather than single genes) play a key role in breast cancer initiation and progression. Our team used Reverse Phase Proteomic Microarray (RPPM) profiling to test for activation of phosphoprotein signaling networks in atypical RPFNA cytology from high-risk premenopausal women in our cohort. RPFNA were obtained from two independent sets of 39 and 38 high-risk premenopausal women; 45% of these women were African American. The signaling network most highly expressed in precancerous cells contained activated signaling proteins associated with the Warburg effect (AKT/mTOR/PI3K), insulin signaling (pACC, IRS1) and epithelial to mesenchymal transition (EMT) IL6/Stat3/vimentin. Conclusions: This is the first evidence that abnormal glucose uptake and the Warburg effect occurs during breast cancer initiation in high-risk African American premenopausal women. These studies demonstrate our ability to identify abnormal glucose and activated signaling networks associated with the Warburg effect in atypical mammary cells from high-risk African American women and provide an important target for breast cancer early detection and prevention. Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):PR2.

The Cyclin-Dependent kinases (CDKs) are a family of essential serine-threonine kinases that are cell cycle regulated by association with cyclins at specific time-points during the cell cycle. Specifically, D-type cyclins form complexes and activate CDK4 and CDK6. We have previously determined that overexpression of CDK4 in mouse epidermis increases malignant progression in chemically induced skin tumors. In order to study the role of CDK6 in squamous epithelial tissues, we generated transgenic mice overexpressing CDK6, driven by the regulatory sequence of the keratin 5 promoter (K5CDK6). Keratinocytes from K5CDK6 mice have similar levels of proliferation compared to wild type mice, however, contrary to the role of CDK4 in mouse skin tumorigenesis, forced expression of CDK6 results in strong reduction of the multiplicity (papillomas per mouse) and incidence of skin tumors. Tumors derived from K5CDK6 or the wild type sibling show no histological differences nor changes in the rate of malignant progression, suggesting that CDK6 expression affects early stages of skin carcinogenesis. Our previous results have also shown that CDK6 preferentially binds to cyclin D3, which overexpression also decreases tumor development in mouse epidermis. Interestingly, lack of Cyclin D1 produces similar phenotype upon chemical carcinogenesis treatment. Thus, we hypothesize that overexpression of Cyclin D3 or CDK6 in a Cyclin D1-null background should synergize with the reduced tumor development previously observed in cyclin D1-null mice. As expected, a lower number of tumors were found in K5-cyclin D3/cyclin D1−/− compound mice compared to cyclin D1−/− siblings. Thus, we hypothesized that the reduced tumor development observed in these mice is a result of the CDK6/Cyclin D3 complex activity. Consequently, K5-CDK6/cyclin D3−/− compound mice are being generated to investigate whether CDK6-associated tumor inhibition depends on cyclin D3 expression. Only in few cases, CDK6 has been associated with tumorigenesis as compared to CDK4 and CDK2, in particular, no association of CDK6 with epidermal tumors has been reported so far. We propose that CDK6/cyclin D3 complexes play an important role in maintaining normal epidermal homeostasis. Therefore we speculate that upregulation of one or both of its components will block tumor development which could be use as a therapeutic approach. Supported by NIH grant CA116328. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3891.

The pRb/Cdk/cyclin/p16 pathway has been found altered in a majority of human neoplasias. In particular the Cyclin-dependent kinase 4 (CDK4) has been found mutated in familial melanoma, amplified or overexpressed in human gliomas, sporadic breast carcinomas and sarcomas. Additionally, a vast number of human cancers contain high levels of CDK4 activity due to loss of the Cdk inhibitor p16Ink4a or overexpression of its binding partner cyclin D1. We have previously demonstrated that CDK4 ablation inhibits chemically-induced mouse skin papillomas, whereas forced expression of CDK4 in mouse epidermis (K5-CDK4) accelerates malignant progression to Squamous Cell Carcinomas (SCC). However, the mechanisms by which changes in CDK4 expression levels control skin tumorigenesis have not been established. In this model topical application of DMBA and TPA results in clonal expansion of a slow cycling stem cell population localized at the bulge area of the hair follicle. Thus, we hypothesize that CDK4 deletion or overexpression affects tumorigenesis by altering the characteristic and/or the number of Keratinocytes Stem Cells (KSC). To address this, we employed the K15-EGFP transgenic mouse model, which expresses EGFP under the control of the keratin 15 promoter in the bulge area of the hair follicle, to generated K15-EGFP/K5-CDK4as well as K15-EGFP/CDK4−/− compound mice. Flow cytometry analysis of K15-EGFP/K5-CDK4 keratinocytes show 4-fold decrease in the number of KSC compared to K15-EGFP control mice. In contrast, K15-EGFP/CDK4−/− compound mice show 4-fold increase in the number of KSC compared with control siblings. Quantification of mRNA in the KSCs showed 3 to 5-fold increase in the CDK4 levels of KSC compared to non-Stem cell population, whereas mild or no changes in CDK2, Cyclin D1 and p27Kip1mRNA levels were detected. Consistent with these data, we also determined an increase number of Labeled-Retaining Cells (LRC) in the hair follicle of CDK4−/− and decreased LRC in K5-CDK4 mice. Together these results are consistent with our hypothesis that changes in CDK4 expression affects the asymmetric cell division of KSC population favoring an increase in the transit amplifying (TA) cell pool and decrease of the KSC pool in K5-CDK4 and the opposite in CDK4−/− mice. As a result, this model predicts that the number of transit amplifying or progenitor cells correlate with the susceptibility to papilloma development. Supported by NIH grant CA116328 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3889.

S-phase kinase-associated protein 2 (Skp2) functions as the receptor component of the Skp-Cullin-F-box complex and is implicated in the degradation of several cell cycle regulators, such as p21(Cip1), p27(Kip1), p57(Kip2), and cyclin E. Numerous studies in human and experimental tumors have demonstrated low p27(Kip1) levels and elevated Skp2 expression. However, a direct association between the inverse correlation of Skp2 and p27(Kip1) with tumorigenesis has not been demonstrated. Herein, we provide evidence that skin tumorigenesis is inhibited in Skp2(-/-) mice. An analysis of mouse keratinocytes indicates that increased p27(Kip1) levels in Skp2(-/-) epidermis cause reduced cell proliferation that is alleviated in the epidermis from Skp2(-/-)/p27(-/-) compound mice. In contrast, we establish that a p27(Kip1) deficiency does not overturn the reduced skin tumorigenesis experienced by Skp2(-/-) mice. In addition, Skp2(-/-) epidermis exhibits an accumulation of p53-cofactor CBP/p300 that is associated with elevated apoptosis in hair follicles and decreased skin tumorigenesis. We conclude that p27(Kip1) accumulation is responsible for the hypoplasia observed in normal tissues of Skp2(-/-) mice but does not have a preponderant function in reducing skin tumorigenesis.