Keywords: breast cancer risk, estrogen withdrawal, KRAS-variant, multiple primary breast cancer, triple negative breast cancer, tumor biologyThe KRAS-variant is a biologically functional, microRNA binding site variant, which predicts increased cancer risk especially for women. Because external exposures, such as chemotherapy, differentially impact the effect of this mutation, we evaluated the association of estrogen exposures, breast cancer (BC) risk and tumor biology in women with the KRAS-variant. Women with BC (n D 1712), the subset with the KRAS-variant (n D 286) and KRAS-variant unaffected controls (n D 80) were evaluated, and hormonal exposures, KRAS-variant status, and pathology were compared. The impact of estrogen withdrawal on transformation of isogenic normal breast cell lines with or without the KRAS-variant was studied. Finally, the association and presentation characteristics of the KRAS-variant and multiple primary breast cancer (MPBC) were evaluated. KRAS-variant BC patients were more likely to have ovarian removal pre-BC diagnosis than non-variant BC patients (p D 0.033). In addition, KRAS-variant BC patients also appeared to have a lower estrogen state than KRAS-variant unaffected controls, with a lower BMI (P < 0.001). Finally, hormone replacement therapy (HRT) discontinuation in KRAS-variant patients was associated with a diagnosis of triple negative BC (P < 0.001). Biologically confirming our clinical findings, acute estrogen withdrawal led to oncogenic transformation in KRAS-variant positive isogenic cell lines. Finally, KRAS-variant BC patients had greater than an 11-fold increased risk of presenting with MPBC compared to non-variant patients (45.39% vs 6.78%,.87], P < 0.001). Thus, estrogen withdrawal and a low estrogen state appear to increase BC risk and to predict aggressive tumor biology in women with the KRAS-variant, who are also significantly more likely to present with multiple primary breast cancer.
Background: The KRAS-variant is a germ-line, microRNA binding site mutation in the KRAS oncogene, which is associated with an increased risk of cancer and altered response to cancer treatment. We investigated the hypothesis that normal cells with the KRAS-variant would be biologically unique. Methods: We created perfectly matched, isogenic normal breast epithelial lines with (MCF10AKRAS+/−, MT) and without (MCF10AKRAS-/−, WT) the KRAS-variant. We also created isogenically matched MCF10A cells with p53 gene-specific stable knockdown with (MLPKRAS+/−) and without (MLPKRAS-/−) the KRAS-variant. We evaluated phenotypic alterations in appearance, gene and microRNA expression and growth in the KRAS-variant vs the parental lines. We used 3-D culture on Matrigel™ (acini formation), and soft agar colony formation (anchorage independent growth) to evaluate growth and transformation. We also investigated EMT and stem cell analysis by performing targeted qRT-PCR, western blotting and flow cytometry. Results: We found that KRAS-variant normal breast epithelial lines (MT) exhibited a mesenchymal phenotype, exhibiting a spindle-like morphology vs the parental (WT) cells, consistent with a baseline epithelial to mesenchymal transition (EMT). In agreement with their appearance, MT cells had significantly lower E-Cadherin and Occludin, and significantly higher Fibronectin and Vimentin than the WT line. We also found that mir-200c was dramatically down-regulated in the MT line, being 828-fold suppressed. miR-200c has been shown to regulate EMT induction and self-renewal and proliferation of stem cells. We thus next investigated whether the MT line had an increased stem cell component. By sorting cells for CD44+/CD24low/− fraction and ALDH1 positivity, we found that MT cells had a significantly higher proportion of CD44+/CD24low/− cells, and higher levels of ALDH1positive cells. In 3-D culture, MT cells formed small irregularly shaped spheroids, whereas WT cells formed well-organized, polarized spheroids, supporting a mesenchymal phenotype. In the MLPKRAS+/− cells we also found a higher stem cell component, however we did not see an EMT phenotype. However, MLPKRAS+/− cells formed disorganized acinar structures, similar to transformed cells, while the MLPKRAS-/− cells formed single, normal round acini. We next plated these cell line in soft agar to test for baseline transformation. While the KRAS-variant normal breast epithelial cells were not transformed at baseline, MLPKRAS+/− cells exhibit colony formation in soft agar, consistent with transformation. Conclusions: These findings indicate that KRAS-variant normal cells are biologically different than cells without the KRAS-variant, but are not transformed. In contrast, p53 suppression in addition to the KRAS-variant is associated with cellular transformation, similar to findings with mutated KRAS. Studies are ongoing to better characterize baseline and oncogenic changes in the presence of the KRAS-variant. Citation Format: Song-Yi Jung, Joanne Weidhaas. Impacts of the KRAS-variant on breast cell biology. [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 1930.
Background: The KRAS-variant is a germ-line, microRNA binding site mutation in the KRAS oncogene, which predicts increased breast cancer risk and aggressive breast tumor biology. We have recently shown clinically that estrogen withdrawal enhances the risk of breast cancer (BC) development and predicts aggressive tumor biology for women with the KRAS-variant. To better understand our clinical results, we have studied the impact of the KRAS-variant and estrogen withdrawal in a newly created isogenically matched normal breast epithelial line, with (MCF10aKRAS+/-; MUT1 and MUT2) versus without (MCF10aKRAS-/-; PAR) the KRAS-variant. Methods: We evaluated phenotypic alterations in appearance, gene and microRNA expression and growth in the lines with the KRAS-variant versus the parental line. We further characterized the impact of estrogen withdrawal on cellular transformation. We used MTT (proliferation), 3-D culture on Matrigel™ (acini formation), and soft agar colony formation (anchorage independent growth) to evaluate growth and transformation differences. We used charcoal stripped media, as well as other clinically used anti-estrogen medications to simulate estrogen withdrawal. We also investigated the underlying molecular changes that lead to transformation in our isogenic MUT cell lines by performing targeted qRT-PCR, western blotting, and Immunocytochemistry. Results: When grown under standard culture conditions, both MUT lines exhibited a fibroblast-like, mesenchymal appearance. Consistent with an epithelial to mesenchymal transition (EMT), the MUT lines showed downregulation of epithelial markers (E-cadherin and Occludin) and upregulation of mesenchymal markers (vimentin, and fibronectin). Furthermore, in 3D culture MUT cells formed small irregularly shaped spheroids, whereas MCF10a PAR cells formed well-organized, polarized spheroids, supporting a mesenchymal phenotype for MUT lines. In anchorage independent growth analysis, MUT1 and MUT 2 cell lines did not form colonies in soft Agar when grown in regular growth culture conditions. However, both MUT cell lines exhibited enhanced transformation with estrogen withdrawal, with a 2-fold increase in colony formation rate in the presence of tamoxifen, a 6.2 fold increase in colony formation rate in charcoal stripped media, and a 7.9 fold increase in colony formation rate with the combination. Conclusions: These findings confirm that normal breast epithelial cells with the KRAS-variant are biologically different than non-variant cells, and that estrogen withdrawal increases their transformation. Studies are ongoing to determine the impact of additional anti-estrogen medications used clinically on the rate of transformation in these lines, as well as to better characterize baseline and oncogenic changes in the presence of the KRAS-variant. Citation Format: Song-Yi Jung, David Salzman, Joanne B. Weidhaas. Estrogen withdrawal and breast cancer development in the presence of the KRAS-variant. [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 2872. doi:10.1158/1538-7445.AM2015-2872
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