Radisky, Derek C.

AbstractBreast cancer recurrence is believed to be caused by a subpopulation of cancer cells that possess the stem cell attribute of treatment resistance. Recently, we and others have reported the generation of breast cancer stem cells (BCSC) by epithelial–mesenchymal transition (EMT), although the physiologic process by which these cells may arise in vivo remains unclear. We show here that exposure of tumor cells to TGFβ and TNFα induces EMT and, more importantly, generates cells with a stable BCSC phenotype which is shown by increased self-renewing capacity, greatly increased tumorigenicity, and increased resistance to oxaliplatin, etoposide, and paclitaxel. Furthermore, gene expression analyses found that the TGFβ/TNFα-derived BCSCs showed downregulated expression of genes encoding claudin 3, 4, and 7 and the luminal marker, cytokeratin 18. These changes indicate a shift to the claudin-low molecular subtype, a recently identified breast cancer subtype characterized by the expression of mesenchymal and stem cell-associated markers and correlated with a poor prognosis. Taken together, the data show that cytokine exposure can be used to generate stable BCSCs ex vivo, and suggest that these cells may provide a valuable tool in the identification of stem cell-directed biomarkers and therapies in breast cancer. Cancer Res; 71(13); 4707–19. ©2011 AACR.

Abstract During development, epithelial cells acquire mesenchymal characteristics through a process known as epithelial-mesenchymal transition (EMT). Substantial evidence now exists that EMT-related processes play a major role in tumor progression. EMT is a focus of interest in lung cancer as loss of E-cadherin expression confers increased resistance to several types of chemotherapy. In previous studies, we found that matrix metalloproteinase-3 (MMP3) induced EMT in mouse mammary epithelial cells through induction of Rac1b, an alternatively spliced isoform of Rac1. We now show that exposure to MMPs stimulates Rac1b and induction of EMT markers in human lung cancer cell lines, and that knockdown of Rac1b blocks MMP induced EMT. We also show that Rac1b expression levels in human lung tumor biopsies is predictive of patient outcome, and that Rac1b and MMP expression levels are correlated in these samples, suggesting a direct relationship. We also describe the generation of new transgenic mouse models for which MMP3 or Rac1b is inducibly expressed in lung epithelial cells; activation of kRAS in these mice reveals that expression of MMP3 or Rac1b promotes increased tumor number, size, and progression to adenocarcinoma as well as increased expression of EMT markers vimentin, sm-actin, col1a1, and Snail. We also show that expression of Rac1b in lung epithelial cells leads to spontaneous adenocarcinoma formation. These results demonstrate a direct linkage between induction of EMT and tumor progression, implicate Rac1b as a key mediator of these processes, and demonstrate that MMPs can directly stimulate all of these events. 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 2318.

Abstract Background: Epithelial proliferation in benign breast disease (BBD) is a well-established biomarker of breast cancer risk, and BBD tissues can be used as a platform to assess other biomarkers for breast cancer risk. Lobular involution (the degree of age-related atrophy in lobules) is a new biomarker, with greater degrees of involution associated with lower breast cancer risk. Successful application of tissue biomarkers of risk to breast tissues from normal women would be valuable for risk prediction in the general population. We hypothesized that normal breast tissue samples would differ from BBD tissues in these histologic features of breast cancer risk, with less epithelial proliferation but a similar degree of involution compared to BBD tissues. Methods: Core biopsy samples from the Mayo Benign Breast Disease Cohort Study were compared to core biopsy samples of normal breast tissue obtained from the Susan G. Komen for the Cure Tissue Bank at IU Simon Cancer Center (KTB), a unique resource of breast tissue from donor women without any known breast abnormalities. All core biopsies were assessed histologically by a single breast pathologist for degree of epithelial proliferation (classified as non-proliferative, proliferative without atypia, or atypical hyperplasia) and extent of lobular involution (classified as none, 1-24%, 25-74%, or >75% of lobules). Data were analyzed using chi-square tests and logistic regression. Results: Overall, 318 Mayo BBD and 303 normal (KTB) core breast biopsy samples were evaluated. As anticipated, significantly less epithelial proliferation was present in normal samples compared to BBD samples. Proliferative disease occurred in 9/303 (3%) of normals versus 79/318 (25%) of BBD, and atypical hyperplasia was seen in 2/303 (0.7%) of normal samples versus 24/318 (8%) of BBD core biopsies, p < 0.0001. This difference remained significant after adjusting for age (which was greater overall in the BBD samples). Complete involution was more frequent in normal compared to BBD tissues, although the difference did not reach statistical significance. The age-adjusted odds ratio of complete involution in normal versus BBD tissues was 1.46 (95% CI 0.98-2.17, p = 0.06), suggesting a possible delay or failure of involution in women with BBD. Conclusions: Histologic features of normal breast tissues are different from those of BBD tissues, with less epithelial proliferation and more lobular involution, consistent with a lower risk of breast cancer expected for women with normal breast tissue compared to those with benign breast disease. Normal breast tissues are a valuable and distinct resource that should be considered for biomarker validation studies. Citation Information: Cancer Prev Res 2010;3(12 Suppl):B23.

Abstract Estrogen receptor (ER) is expressed in normal and malignant breast epithelium, and expression levels have been found to increase with age in normal breast epithelium but not in atypical hyperplasia (AH) and carcinoma in situ. Here we assess ER expression in AH and its association with later breast cancer. ER expression was assessed immunohistochemically in archival sections from 246 women with AH who had open benign breast biopsy from 1967 to 1991. The ACIS III (Dako) was utilized to calculate ER expression in all atypical foci. Using multivariate linear regression, we examined associations of ER expression with age at biopsy, indication for biopsy, type of atypia, number of atypical foci, involution status, and family history. Breast cancer risk across levels of ER expression was also assessed compared with the Iowa SEER control population. Among 246 women, 87 (35%) had atypical ductal hyperplasia (ADH), 141 (57%) had atypical lobular hyperplasia (ALH), and 18 (7%) had both. Forty-nine (20%) developed breast cancer (median follow-up of 14.4 years). Multivariate analysis indicated that type of atypia and age at diagnosis were significantly associated with ER percent staining and intensity (P < 0.05). ER expression was increased in women with ADH and/or those over age 55. ER expression did not significantly impact breast cancer risk in patients diagnosed with atypia. We found increasing ER expression in AH with increasing age. ER expression in AH does not further discriminate breast cancer risk in women with atypia. Cancer Prev Res; 4(3); 435–44. ©2011 AACR.

Abstract Elements of the immune system act as intimate regulators of cancer progression, inhibiting early stages of tumor growth, through immunosurveillance while facilitating later stages of tumor progression. Recent findings have revealed that activated CD8 T cells can stimulate mammary epithelial tumor cells to undergo epithelial-mesenchymal transition (EMT) and to acquire the greatly increased tumorigenic capability and chemotherapeutic resistance of breast cancer stem cells (BCSC). These studies provide a window to understanding how BCSC arise and are maintained within tumors, and how to best target these processes for therapeutic benefit. Cancer Res; 70(8); 3005–8. ©2010 AACR.

Purpose Mammographic density and lobular involution are both significant risk factors for breast cancer, but whether these reflect the same biology is unknown. We examined the involution and density association in a large benign breast disease (BBD) cohort. Patients and Methods Women in the Mayo Clinic BBD cohort who had a mammogram within 6 months of BBD diagnosis were eligible. The proportion of normal lobules that were involuted was categorized by an expert pathologist as no (0%), partial (1% to 74%), or complete involution (≥ 75%). Mammographic density was estimated as the four-category parenchymal pattern. Statistical analyses adjusted for potential confounders and evaluated modification by parity and age. We corroborated findings in a sample of women with BBD from the Mayo Mammography Health Study (MMHS) with quantitative percent density (PD) and absolute dense and nondense area estimates. Results Women in the Mayo BBD cohort (n = 2,667) with no (odds ratio, 1.7; 95% CI, 1.2 to 2.3) or partial (odds ratio, 1.3; 95% CI, 1.0 to 1.6) involution had greater odds of high density (DY pattern) than those with complete involution (P trend < .01). There was no evidence for effect modification by age or parity. Among 317 women with BBD in the MMHS study, there was an inverse association between involution and PD (mean PD, 22.4%, 21.6%, 17.2%, for no, partial, and complete, respectively; P trend = .04) and a strong positive association of involution with nondense area (P trend < .01). No association was seen between involution and dense area (P trend = .56). Conclusion We present evidence of an inverse association between involution and mammographic density.

Purpose Accurate, individualized risk prediction for breast cancer is lacking. Tissue-based features may help to stratify women into different risk levels. Breast lobules are the anatomic sites of origin of breast cancer. As women age, these lobular structures should regress, which results in reduced breast cancer risk. However, this does not occur in all women. Methods We have quantified the extent of lobule regression on a benign breast biopsy in 85 patients who developed breast cancer and 142 age-matched controls from the Mayo Benign Breast Disease Cohort, by determining number of acini per lobule and lobular area. We also calculated Gail model 5-year predicted risks for these women. Results There is a step-wise increase in breast cancer risk with increasing numbers of acini per lobule (P = .0004). Adjusting for Gail model score, parity, histology, and family history did not attenuate this association. Lobular area was similarly associated with risk. The Gail model estimates were associated with risk of breast cancer (P = .03). We examined the individual accuracy of these measures using the concordance (c) statistic. The Gail model c statistic was 0.60 (95% CI, 0.50 to 0.70); the acinar count c statistic was 0.65 (95% CI, 0.54 to 0.75). Combining acinar count and lobular area, the c statistic was 0.68 (95% CI, 0.58 to 0.78). Adding the Gail model to these measures did not improve the c statistic. Conclusion Novel, tissue-based features that reflect the status of a woman's normal breast lobules are associated with breast cancer risk. These features may offer a novel strategy for risk prediction.

Abstract The breast cancer stem cell (BCSC) hypotheses suggest that breast cancer is derived from a single tumor-initiating cell with stem-like properties, but the source of these cells is unclear. We previously observed that induction of an immune response against an epithelial breast cancer led in vivo to the T-cell–dependent outgrowth of a tumor, the cells of which had undergone epithelial to mesenchymal transition (EMT). The resulting mesenchymal tumor cells had a CD24−/loCD44+ phenotype, consistent with BCSCs. In the present study, we found that EMT was induced by CD8 T cells and the resulting tumors had characteristics of BCSCs, including potent tumorigenicity, ability to reestablish an epithelial tumor, and enhanced resistance to drugs and radiation. In contrast to the hierarchal cancer stem cell hypothesis, which suggests that breast cancer arises from the transformation of a resident tissue stem cell, our results show that EMT can produce the BCSC phenotype. These findings have several important implications related to disease progression and relapse. [Cancer Res 2009;69(7):2887–95]

Abstract Anaplastic thyroid carcinoma (ATC) is a highly aggressive form of the disease for which new therapeutic options are desperately needed. Previously, we showed that the high-affinity peroxisome proliferator–activated receptor γ (PPARγ) agonist, RS5444, inhibits cell proliferation of ATC cells via induction of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 (p21). We show here that up-regulation of RhoB is a critical step in PPARγ-mediated activation of p21-induced cell stasis. Using multiple independently derived ATC cell lines, we found that treatment with RS5444 leads to the up-regulation of RhoB and subsequent activation of p21, and that silencing of RhoB by RNAi blocks the ability of RS5444 to induce p21 and to inhibit cell proliferation. Our results show that transcriptional regulation of RhoB by the nuclear transcription factor PPARγ is responsible for the induction of p21 mRNA and protein. We further implicate RhoB as a key signaling effector for the growth inhibition of ATC, as treatment with a histone deacetylase inhibitor shown to increase RhoB expression in lung cancer cells caused the up-regulation of RhoB in ATC cells accompanied by increased expression of p21 and inhibition of cell proliferation; this effect occurred even in ATC cells that were unresponsive to RS5444 due to a lack of expression of PPARγ. Our results implicate RhoB as a novel intermediate in critical signaling pathways and as an additional target for therapeutic intervention in ATC. [Cancer Res 2009;69(4):1536–44]

Abstract Expression of neuropilin-1 (NRP-1) has been shown in many cancer cells, but its molecular effect on tumorigenesis is largely unknown. In this report, we show that in aggressive types of renal cell carcinoma (RCC), NRP-1 is expressed at a high level. We show that after knockdown of NRP-1 by short hairpin RNA, RCC cells express significantly lower levels of MDM-2 and p63 proteins but higher levels of p53, and exhibit reduced migration and invasion. When implanted in mice, RCC cells with a reduced NRP-1 level have a statistically significant smaller tumor-forming ability than control cells. Also, NRP-1 knockdown RCC cells exhibit a more differentiated phenotype, as evidenced by the expression of epithelial-specific and kidney-specific cadherins, and the inhibition of sonic hedgehog expression participated in this effect. Inhibition of sonic hedgehog expression can be reversed by ΔNp63α overexpression. Our study reveals that NRP-1 helps maintain an undifferentiated phenotype in cancer cells. [Cancer Res 2008;68(21):8667–72]