Therese Cusick

Expression profiling of in vivo ductal carcinoma in situ progression models identified B cell lymphoma-9 as a molecular driver of breast cancer invasion

IntroductionThere are an estimated 60,000 new cases of ductal carcinoma in situ (DCIS) each year. A lack of understanding in DCIS pathobiology has led to overtreatment of more than half of patients. We profiled the temporal molecular changes during DCIS transition to invasive ductal carcinoma (IDC) using in vivo DCIS progression models. These studies identified B cell lymphoma-9 (BCL9) as a potential molecular driver of early invasion. BCL9 is a newly found co-activator of Wnt-stimulated β-catenin-mediated transcription. BCL9 has been shown to promote progression of multiple myeloma and colon carcinoma. However BCL9 role in breast cancer had not been previously recognized.MethodsMicroarray and RNA sequencing were utilized to characterize the sequential changes in mRNA expression during DCIS invasive transition. BCL9-shRNA knockdown was performed to assess the role of BCL9 in in vivo invasion, epithelial-mesenchymal transition (EMT) and canonical Wnt-signaling. Immunofluorescence of 28 patient samples was used to assess a correlation between the expression of BCL9 and biomarkers of high risk DCIS. The cancer genome atlas data were analyzed to assess the status of BCL9 gene alterations in breast cancers.ResultsAnalysis of BCL9, by RNA and protein showed BCL9 up-regulation to be associated with DCIS transition to IDC. Analysis of patient DCIS revealed a significant correlation between high nuclear BCL9 and pathologic characteristics associated with DCIS recurrence: Estrogen receptor (ER) and progesterone receptor (PR) negative, high nuclear grade, and high human epidermal growth factor receptor2 (HER2). In vivo silencing of BCL9 resulted in the inhibition of DCIS invasion and reversal of EMT. Analysis of the TCGA data showed BCL9 to be altered in 26 % of breast cancers. This is a significant alteration when compared to HER2 (ERBB2) gene (19 %) and estrogen receptor (ESR1) gene (8 %). A significantly higher proportion of basal like invasive breast cancers compared to luminal breast cancers showed BCL9 amplification.ConclusionBCL9 is a molecular driver of DCIS invasive progression and may predispose to the development of basal like invasive breast cancers. As such, BCL9 has the potential to serve as a biomarker of high risk DCIS and as a therapeutic target for prevention of IDC.

An evaluation of resident training in breast procedures

BACKGROUND The purpose of this study was to determine if senior residents are participating in advanced level breast cases. METHODS A retrospective review of 1,583 breast procedures on 1,213 patients over 2 years was performed. Each case was evaluated for the type of case: (1) junior level (PGY 1 and 2)--core needle biopsy, fine needle aspirate, excisional biopsy, needle localized biopsy, lumpectomy, partial mastectomy, and simple mastectomy; and (2) senior level--stereotactic breast biopsy, sentinel lymph node biopsy, axillary dissection, and modified radical mastectomy. All cases were reviewed for level of resident involved. RESULTS Overall, seniors performed 31% of the cases, junior residents performed 42%, and 27% were unattended by any resident. Senior level breast cases were specifically examined to compare whether a senior or junior was present. Overall, 891 defined senior cases were performed. A senior level resident attended 34% of these, leaving 66% covered by junior residents or uncovered altogether. CONCLUSIONS It is clear when evaluating breast procedures that senior residents are less involved than junior residents. Furthermore, the fact that many of the reported cases (28%) and, more significantly, senior level cases (26%) have no resident involved leads us to conclude that senior level residents are not taking advantage of the opportunity to gain experience in performing advanced level breast procedures in our surgical program.

NEMO, a transcriptional target of estrogen and progesterone, is linked to tumor suppressor PML in breast cancer

The beneficial versus detrimental roles of estrogen plus progesterone (E+P) in breast cancer remains controversial. Here we report a beneficial mechanism of E+P treatment in breast cancer cells driven by transcriptional upregulation of the NFκB modulator NEMO, which in turn promotes expression of the tumor suppressor protein promyelocytic leukemia (PML). E+P treatment of patient-derived epithelial cells derived from ductal carcinoma in situ (DCIS) increased secretion of the proinflammatory cytokine IL6. Mechanistic investigations indicated that IL6 upregulation occurred as a result of transcriptional upregulation of NEMO, the gene that harbored estrogen receptor (ER) binding sites within its promoter. Accordingly, E+P treatment of breast cancer cells increased ER binding to the NEMO promoter, thereby increasing NEMO expression, NFκB activation, and IL6 secretion. In two mouse xenograft models of DCIS, we found that RNAi-mediated silencing of NEMO increased tumor invasion and progression. This seemingly paradoxical result was linked to NEMO-mediated regulation of NFκB and IL6 secretion, increased phosphorylation of STAT3 on Ser727, and increased expression of PML, a STAT3 transcriptional target. In identifying NEMO as a pivotal transcriptional target of E+P signaling in breast cancer cells, our work offers a mechanistic explanation for the paradoxical antitumorigenic roles of E+P in breast cancer by showing how it upregulates the tumor suppressor protein PML. Cancer Res; 77(14); 3802-13. ©2017 AACR.

Abstract 1853: Ovarian steroid hormones promote progression of DCIS by increasing cancer stem cell self-renewal through IL-6 signaling

Introduction: The ability of the ovarian steroid hormones estradiol (E) and progesterone (P) to influence cancer stem cell (CSC) self-renewal and progression of human ductal carcinoma in situ (DCIS) is a critical but understudied area or research. The objective of this study is to characterize steroid hormone signaling involved in CSC self-renewal and invasion in ER+PR+ DCIS. We have found that these hormones can increase CSC self-renewal in a subset of DCIS patients, and that this may be facilitated by activation of the IL-6 pathway. Methods: Mammosphere (MS) assays (an in vitro assessment of CSC self-renewal) and the mouse intraductal (MIND) xenograft model were used to characterize hormonal regulation of the CSC population and invasion potential of primary human ER+PR+ DCIS cells. Additionally, ovarectomized mice were used to create MIND xenografts of ER+PR+ DCIS, treated with E+P or vehicle for 8 weeks, and used for RNA sequencing and immunofluorescent staining. Results: Only a subset of cases responded to E+P in vitro by increasing MS efficiency, indicating increased CSC self-renewal. RNA sequencing of MSs identified genes that differed between vehicle and E+P treatment only in DCIS cases that respond to E+P by increasing MS efficiency. Many of these changes were associated with epithelial mesenchymal transition (EMT), including Twist1, Snai1, MMP9. In E+P-treated MIND xenografts, DCIS cases that respond to E+P by increasing MS efficiency also had higher IL-8 and vimentin (markers of EMT) compared to non-responders (P Conclusions: Our results indicate that steroid hormone treatment can enrich the CSC population by increasing MS efficiency in some but not all DCIS cases, and that IL-6 may be involved in this selective ability. Therefore, DCIS cases with higher MS efficiency in response to E+P treatment may also show higher invasion potential in vivo. Our current model is that steroid hormones induce increased secretion of IL-6, leading to increased self-renewal and invasion potential. Further characterization of the signaling pathway(s) associated with this selective effect may allow us to predict which DCIS lesions have the ability to progress to invasive ductal carcinoma and allow development of novel therapeutic targets. Citation Format: Kelli E. Valdez, Yan Hong, Fang Fan, Lisa May, Therese Cusick, Marc Inciardi, Mark Redick, Jason Gatewood, Fariba Behbod. Ovarian steroid hormones promote progression of DCIS by increasing cancer stem cell self-renewal through IL-6 signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1853. doi:10.1158/1538-7445.AM2015-1853

Abstract B038: Ovarian steroid hormones regulate cancer stem cell self-renewal through aberrant signaling pathways in primary human ductal carcinoma in situ

Introduction: The ability of the ovarian steroid hormones estradiol and progesterone to influence mammary stem cell self-renewal has been demonstrated in several prominent studies using models of both normal and pathological mammary gland growth. The common theme appears to involve estradiol and progesterone acting on the ER, PR-expressing luminal cells, which in turn through paracrine signaling can induce proliferation and increased self-renewal of both normal and cancer stem cells (CSC). However, none of these studies have examined the ability of these steroid hormones to influence CSC self-renewal and progression of ductal carcinoma in situ (DCIS). Purpose: The objective of this study is to characterize steroid hormone paracrine signaling involved in aberrant stem cell self-renewal and invasion in human primary DCIS. Our hypothesis is that those DCIS cases which respond to hormonal treatment by increasing CSC self-renewal also have the greatest invasive potential and risk of malignant progression. Experimental design: Mammosphere assays, an in vitro assessment of self-renewal, and the in vivo mouse intraductal (MIND) xenograft model are being used to characterize hormonal regulation of the stem cell population and invasion potential of primary human DCIS cells obtained from patient biopsies. Results: Cells from primary human DCIS, atypical hyperplasia, and DCIS with microinvasion were cultured under non-adherent conditions to allow primary mammosphere (MS) formation in the presence of vehicle, 10 nM estradiol, 100 nM progesterone, or both. After 14 days, cells were passaged to allow secondary MS formation. We found that only a subset of cases responded to the steroid hormone treatment by increasing MS forming efficiency, indicating increased self-renewal ability. Using human growth factor antibody arrays to assay the cell culture media, we found that insulin-like growth factor 2 (IGF2) was selectively up-regulated in response to steroid hormone treatment only in those cases which also demonstrated increased CSC self-renewal. Changes in gene expression in response to treatment are also being examined by next generation RNA sequencing. RNA from vehicle and steroid-treated DCIS cells was isolated from cases that either did or did not respond to E+P treatment. Differential expression analysis was used to identify genes that differed between vehicle and E+P treatment only in DCIS cases that respond to E+P by increasing mammosphere-forming efficiency. Ingenuity Pathway Analysis was used to characterize the biological functions and disease states associated with gene changes observed. The top results included cellular movement, cancer, and reproductive system disease. Many of the gene changes observed were associated with changes that appear during epithelial mesenchymal transition (EMT). This supports our hypothesis that the cases which respond to steroid hormone treatment by increasing stem cell self- renewal may have greater invasive potential. Characterization of MIND xenografts created using cells from these same cases will allow us to determine if these cases also have greater growth and invasion potential in vivo. Conclusions: Our results indicate that steroid hormone treatment can enrich the stem cell population by increasing MS efficiency in some but not all DCIS cases, and that IGF2 may be involved in this selective ability. Based on these data, we expect that DCIS cases with higher MS efficiency in response to steroid hormone treatment will also show higher invasion potential in vivo and in vitro. Further characterization of the signaling pathway(s) associated with this selective effect may allow us to predict which DCIS lesions have the ability to progress to invasive ductal carcinoma. Citation Format: Kelli E. Valdez, Yan Hong, Fang Fan, Therese Cusick, Lisa S. May, William P. Smith, Fariba Behbod. Ovarian steroid hormones regulate cancer stem cell self-renewal through aberrant signaling pathways in primary human ductal carcinoma in situ. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B038.

Abstract C41: Ovarian steroid hormones influence progression of premalignant breast cancer by disregulated cancer stem cell self-renewal

Introduction: Ductal carcinoma in situ (DCIS), the premalignant form of human breast cancer, only progresses to invasive breast cancer (IBC) in an estimated 50% of diagnosed cases. Elucidation of the mechanisms underlying this selective event could prevent unnecessary treatment and increase the accuracy of diagnosis. Progression to an invasive phenotype has been associated with an increase in mammary cancer stem cells (CSCs). The ability of the ovarian steroid hormones estradiol and progesterone to influence mammary stem cell self-renewal has been demonstrated in models of both normal and pathological mammary gland growth. The common theme appears to involve estradiol and progesterone acting on the ER, PR-expressing luminal cells, which in turn through paracrine signaling can induce proliferation and increased self-renewal of both normal and CSC. However, none of these studies have examined the ability of these steroid hormones to influence CSC self-renewal and progression of DCIS. Purpose: The objective of this study is to characterize steroid hormone paracrine signaling involved in aberrant stem cell self-renewal and invasion in human primary DCIS. Our hypothesis is that those DCIS cases which respond to hormonal treatment by increasing CSC self-renewal also have the greatest invasive potential and risk of malignant progression. Experimental design: Mammosphere assays, an in vitro assessment of self-renewal, and the in vivo mouse intraductal (MIND) xenograft model are being used to characterize hormonal regulation of the stem cell population and invasion potential of primary human DCIS cells obtained from patient biopsies. Results: Cells from primary human DCIS, atypical hyperplasia, and DCIS with microinvasion were cultured under non-adherent conditions to allow primary mammosphere (MS) formation in the presence of vehicle, 10 nM estradiol, 100 nM progesterone, or both. After 14 days, cells were passaged to allow secondary MS formation. We found that only a subset of cases responded to the steroid hormone treatment by increasing MS forming efficiency, indicating increased self-renewal ability. Using human growth factor antibody arrays to assay the cell culture media, we found that insulin-like growth factor 2 (IGF2) was selectively up-regulated in response to steroid hormone treatment only in those cases which also demonstrated increased CSC self-renewal. Changes in gene expression in response to treatment are also being examined by next generation RNA sequencing. RNA from vehicle and steroid-treated DCIS cells was isolated from cases that either did or did not respond to E+P treatment. Differential expression analysis was used to identify genes that differed between vehicle and E+P treatment only in DCIS cases that respond to E+P by increasing mammosphere-forming efficiency. Ingenuity Pathway Analysis was used to characterize the biological functions and disease states associated with gene changes observed. The top results included cellular movement, cancer, and reproductive system disease. Many of the gene changes observed were associated with changes that appear during epithelial mesenchymal transition (EMT). This supports our hypothesis that the cases which respond to steroid hormone treatment by increasing stem cell self- renewal may have greater invasive potential. Additionally, MIND xenografts in ovariectomized mice are being created and treated with steroid hormones using cells from these same DCIS cases to determine if these cases also have greater growth and invasion potential in vivo. Differences in gene expression of Wnt4, RankL, FGF9, IGF2, and amphiregulin will also be examined, as these have been shown to be involved in the signaling pathways regulating steroid hormone-induced stem cell proliferation. Conclusions: Our results indicate that steroid hormone treatment can enrich the stem cell population by increasing MS efficiency in some but not all DCIS cases, and that IGF2 may be involved in this selective ability. Based on these data, we expect that DCIS cases with higher MS efficiency in response to steroid hormone treatment will also show higher invasion potential in vivo and in vitro. Further characterization of the signaling pathway(s) associated with this selective effect may allow us to predict which DCIS lesions have the ability to progress to invasive ductal carcinoma. Citation Format: Kelli E. Valdez, Yan Hong, Fang Fan, Therese Cusick, Lisa May, William Smith, Fariba Behbod. Ovarian steroid hormones influence progression of premalignant breast cancer by disregulated cancer stem cell self-renewal. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr C41.

Evaluation of changes in breast architecture after preoperative chemotherapy for breast cancer

BACKGROUND Although use of preoperative chemotherapy for breast cancer is increasing, resultant changes in breast architecture have not been described. The purpose of this study was to examine breast architecture changes in response to chemotherapy by the placement of 4 peripheral clips. METHODS In a prospective case-series of breast cancer patients selected to undergo preoperative chemotherapy, 4 clips were placed peripherally to each mass using sonographic guidance. Mammograms documented tumor size and clip locations both before chemotherapy and after chemotherapy. Percentage reduction in area was calculated based on the tumor dimensions and distances between clips. RESULTS In 16 participants, 87.5% of lesions had a significant response to chemotherapy. Changes in clip measurements varied widely from significant reduction to significant increase and did not correlate with changes in tumor size. The Pearson correlation coefficient comparing changes in tumor size and clip measurements was .036 (P = .895). CONCLUSIONS There was no correlation between reduction in tumor size and change in clip measurements. Further research should be conducted using noncompression breast imaging modalities to eliminate possible distortion caused by mammographic compression.