Radisky, Derek C.

Abstract Breast tumors are stiff and hypoxic. Nevertheless, it remains unclear how stiff and hypoxic conditions within the tumor microenvironment promote breast cancer progression. Using an innovative engineered culture model to recapitulate these features, we found that, under stiff and hypoxic conditions, breast cancer cells have enhanced integrin-linked kinase (ILK) expression and acquire cancer stem cell (CSC)-like traits, suggesting tissue stiffness and oxygen tension can modulate ILK expression to induce breast CSC formation. Knocking down ILK impairs the mechanosensing of invasive breast cancer cells on stiff microenvironments, inhibits CSC markers and properties. In contrast, ectopic expression of ILK promotes breast CSC formation. In addition to promoting CSC-like phenotype, microarray analysis reveals that stiff and hypoxic microenvironments also regulate genes involved in mRNA processing, splicing and the spliceosome. These data suggest that the non-cellular compartment of the tumor microenvironment, namely tissue mechanics and oxygen tension, can promote breast cancer progression by controlling mechanotransduction and post-transcriptional regulation of breast cancer cells. Note: This abstract was not presented at the meeting. Citation Format: Mei-Fong Pang, Derek C. Radisky, Celeste M. Nelson. Role of tissue stiffness and oxygen tension in promoting breast cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5943. doi:10.1158/1538-7445.AM2017-5943

Abstract This study investigates how increased stiffness of the tumor microenvironment can induce cellular multinucleation, an easily observable marker of polyploidy. Up to 37% percent of tumors exhibit whole-genome doubling, which typically precedes other somatic copy number alterations. Additionally, induction of tetraploidy in human cells promotes increased tolerance for mutation, resistance to chemotherapeutic drugs, and transformation in culture. Tumors are inherently stiffer than normal tissue, and this property has been shown to affect cell growth and proliferation. Similarly, cell cycle errors have long been linked to chromosomal abnormalities. Here, we used engineered two-dimensional substrata that mimic the stiffness of tumor and normal microenvironments to investigate how matrix stiffness regulates multinucleation in mammary epithelial cells. Multinucleation was quantified by staining with Hoescht to visualize the nuclei. Timelapse microscopy enabled visualization of the process by which cells become multinucleated. Changes in gene expression were determined by quantitative RT-PCR. Cells cultured on “stiff” substrata, representing tumor tissue, showed a nearly 14-fold increase in multinucleation compared to cells cultured on “soft” substrata, representing normal tissue. We found that multinucleation was regulated in part by signaling downstream of matrix metalloproteinase-3 (MMP3), which is commonly upregulated in cancer and known to induce epithelial-mesenchymal transition (EMT). This signaling depended on expression of the Rac1 splice variant, Rac1b, production of ROS, and expression of Snail. Under all conditions, cells cultured on soft substrata maintained a low frequency of multinucleation. Multinucleation on stiff substrata primarily resulted from midbody abscission failure. A soft microenvironment protected the stability of the genome in epithelial cells by preventing midbody stability, which depended on septin 4, a novel target of Snail. Importantly, we found that transforming growth factor-β (TGFβ), another EMT-inducer, also caused multinucleation downstream of Snail, which was prevented by culture on soft substrata. Our data thus suggest that tissue stiffening during tumorigenesis synergizes with oncogenic signaling to promote genomic abnormalities that drive cancer progression. Further, our results suggest that EMT-related signaling pathways are associated with disease progression not necessarily because they induce metastasis, but because they induce genomic instability. Note: This abstract was not presented at the meeting. Citation Format: Allison K. Simi, Alisya A. Anlas, Sherry X. Zhang, Tiffaney Hsia, Derek C. Radisky, Celeste M. Nelson. A soft microenvironment protects from failure of midbody abscission and multinucleation downstream of EMT initiators [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5914. doi:10.1158/1538-7445.AM2017-5914

ABSTRACTMatrix metalloproteinases (MMPs) are a family of zinc endopeptidases that cleave nearly all components of the extracellular matrix as well as many other soluble and cell‐associated proteins. MMPs have been implicated in normal physiological processes, including development, and in the acquisition and progression of the malignant phenotype. Disappointing results from a series of clinical trials testing small molecule, broad spectrum MMP inhibitors as cancer therapeutics led to a re‐evaluation of how MMPs function in the tumor microenvironment, and ongoing research continues to reveal that these proteins play complex roles in cancer development and progression. It is now clear that effective targeting of MMPs for therapeutic benefit will require selective inhibition of specific MMPs. Here, we provide an overview of the MMP family and its biological regulators, the tissue inhibitors of metalloproteinases (TIMPs). We then summarize recent research from model systems that elucidate how specific MMPs drive the malignant phenotype of breast cancer cells, including acquisition of cancer stem cell features and induction of the epithelial–mesenchymal transition, and we also outline clinical studies that implicate specific MMPs in breast cancer outcomes. We conclude by discussing ongoing strategies for development of inhibitors with therapeutic potential that are capable of selectively targeting the MMPs most responsible for tumor promotion, with special consideration of the potential of biologics including antibodies and engineered proteins based on the TIMP scaffold. J. Cell. Biochem. 118: 3531–3548, 2017. © 2017 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.

2 Background: Women with atypical hyperplasia (AH) on breast biopsy have an aggregate increased risk of breast cancer (BC), but accurate personalized risk prediction is desirable to facilitate individual clinical management decisions. Currently used models provide poor BC risk prediction for women with AH. Our goal was to develop and validate an improved risk prediction model for women with AH. Methods: From a cohort of 13,538 women with benign breast disease from 1967-2001, pathology review confirmed 699 with AH. Clinical risk factors and histologic features of the tissue biopsy were recorded, and BC events were ascertained from study questionnaires, tumor registry, and review of medical records. Using a lasso approach, 23 variables were assessed for model inclusion. Lasso-identified features were then fit in a Cox regression model to estimate BC risk. Model discrimination was assessed with C-statistics in the model-building set and in a separate external validation set. Calibration was assessed by comparing observed to predicted breast cancer counts. Results: The model-building set comprised 699 women with 142 BC events (median follow-up 8.1 years), and the external validation set comprised 461 women with 114 BC events (median follow-up 11.4 years). The final model included three covariates: age at biopsy, age squared, and number of foci of AH. Model performance was good, with a C-statistic of 0.622 (SE = 0.027) in the model-building set and 0.594 (SE = 0.029) in the external validation set. The model is well-calibrated, with observed to expected numbers of BCs nearly equal across all post-biopsy follow-up years. Conclusions: We propose a new model for predicting BC risk in women with AH based on age at biopsy and number of foci of atypia. This model provides absolute risk estimates for women with AH, has good discriminatory ability, is well-calibrated, and validates in an external cohort.

18 Background: Benign breast disease (BBD) is diagnosed in 1-2 million women/year in the US, and while these patients are known to be at substantially increased risk for subsequent development of breast cancer, existing models for risk assessment perform poorly at the individual level. Here, we describe a DNA-microarray-based transcriptional model for breast cancer risk prediction for patients with sclerosing adenosis (SA), a lesion found in ¼ of all BBD patients that is characterized by epithelial proliferation, disordered acinar architecture, and stromal fibrosis. Methods: A training set was developed from 86 patients diagnosed with sclerosing adenosis (SA), of which 27 subsequently developed cancer within 10 years (cases) and 59 remained cancer-free at 10 years (controls). A diagonal linear discriminate analysis (DLDA)-prediction model for prediction of cancer within 10 years (SA TTC10) was generated from transcriptional profiles of formalin fixed, paraffin-embedded (FFPE) biopsy-derived RNA. This model was tested on a separate validation case-control set composed of 65 SA patients. Results: The SA TTC10 gene signature model, composed of 35 gene features, achieved a clear and significant separation between case and control with receiver operating characteristic area under the curve of 0.913 in the training set and 0.836 in the validation set. Conclusions: Our results provide the first demonstration that benign breast tissue contains transcriptional alterations that indicate risk of breast cancer development, and that essential precursor biomarkers of malignancy are present many years prior to cancer development. Furthermore, the SA TTC10 gene signature model, which can be assessed on FFPE biopsies, constitutes a novel prognostic biomarker for patients with SA.

Abstract Purpose: Little is known about the role of the immune system in the earliest stages of breast carcinogenesis. We studied quantitative differences in immune cell types between breast tissues from normal donors and those from women with benign breast disease (BBD). Experimental Design: A breast tissue matched case–control study was created from donors to the Susan G. Komen for the Cure Tissue Bank (KTB) and from women diagnosed with BBD at Mayo Clinic (Rochester, MN) who either subsequently developed cancer (BBD cases) or remained cancer-free (BBD controls). Serial tissue sections underwent immunostaining and digital quantification of cell number per mm2 for CD4+ T cells, CD8+ T cells, CD20+ B cells, and CD68+ macrophages and quantification of positive pixel measure for CD11c (dendritic cells). Results: In 94 age-matched triplets, BBD lobules showed greater densities of CD8+ T cells, CD11c+ dendritic cells, CD20+ B cells, and CD68+ macrophages compared with KTB normals. Relative to BBD controls, BBD cases had lower CD20+ cell density (P = 0.04). Nearly 42% of BBD cases had no CD20+ B cells in evaluated lobules compared with 28% of BBD controls (P = 0.02). The absence of CD20+ cells versus the presence in all lobules showed an adjusted OR of 5.7 (95% confidence interval, 1.4–23.1) for subsequent breast cancer risk. Conclusions: Elevated infiltration of both innate and adaptive immune effectors in BBD tissues suggests an immunogenic microenvironment. The reduced B-cell infiltration in women with later breast cancer suggests a role for B cells in preventing disease progression and as a possible biomarker for breast cancer risk. Clin Cancer Res; 23(14); 3945–52. ©2017 AACR.

Abstract We have investigated the distribution of PDGFR-a and -b in the stroma of normal breast tissue, ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) by immunohistochemistry. Both receptors were expressed by fibroblasts of normal breast tissue. While the PDGFRb remained high during cancer progression, the PDGFRa was lost in most of the DCIS samples and not detectable in IDC. The patient group with PDGFRa(low)/PDGFRb(high) fibroblasts had the highest risk for local recurrences or metastasis. The characteristic loss of stromal PDGFRa expression was also observed in the PyMT mouse breast cancer model. In vitro co-culture experiments confirmed a tumor cell-induced differentiation of fibroblasts, resulting in a fibroblast population marked by PDGFRa(low)/PDGFRb(high) expression. We could identify the interaction of Jagged-1 expressed by the tumor cells with Notch-2 expressed on the fibroblasts, as an essential activator of TGFb signaling in fibroblasts. Gene expression analysis and reporter-gene assays of sorted cells from co-cultures revealed, that fibroblasts switch from a more anti-tumorigenic signaling (expression of BMPs and Wnt antagonists) towards a pro-tumorigenic signaling (expression of BMP inhibitors, TGFb and Wnt ligands). Co-cultured tumor cells show an increased migratory activity and the induction of a mesenchymal phenotype. Inhibition of Notch or TGF signaling in fibroblasts could block these phenotypes. To investigate the impact of fibroblasts on the progression of DCIS to IDC, a DCIS xenograft model was used. Co-injection of MCF10.DCIS cells with human breast fibroblasts, expressing both PDGFRs, into the mouse mammary fat pad resulted in an earlier tumor take with faster tumor growth. The corresponding co-injection experiments with fibroblast and tumor cells, that were depleted of Notch2 respectively Jagged-1 expression by CRISPR/CAS technology is currently ongoing. Collectively the study identifies a potentially druggable paracrine cross-talk between epithelial and stromal cells, which promotes DCIS progression and involves a Notch mediated up-regulation of TGFb signaling. The study also suggests that loss of PDGFRa marks a subset of fibroblasts with specific tumor-promoting activities. Citation Format: Carina Strell, Janna Paulsson, Artur Mezheyeuski, Shao-Bo Jin, Pernilla Roswall, Johanna Andrea, Oliver Frings, Fredrick Warnberg, Hemming Johansson, Karin Jirstrom, Kristian Pietras, Per Hall, Urban Lendahl, Christer Betsholtz, Derek C. Radisky, Arne Ostman. Cell-contact dependent epithelial-stromal cross talk, including a modulation of stromal PDGFR expression, drives the progression of early-stage breast cancer lesions. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B41.

Abstract Breast tumors are stiffer and more hypoxic than nonmalignant breast tissue. Here we report that stiff and hypoxic microenvironments promote the development of breast cancer stem-like cells (CSC) through modulation of the integrin-linked kinase ILK. Depleting ILK blocked stiffness and hypoxia-dependent acquisition of CSC marker expression and behavior, whereas ectopic expression of ILK stimulated CSC development under softer or normoxic conditions. Stiff microenvironments also promoted tumor formation and metastasis in ovo, where depleting ILK significantly abrogated the tumorigenic and metastatic potential of invasive breast cancer cells. We further found that the ILK-mediated phenotypes induced by stiff and hypoxic microenvironments are regulated by PI3K/Akt. Analysis of human breast cancer specimens revealed an association between substratum stiffness, ILK, and CSC markers, insofar as ILK and CD44 were expressed in cancer cells located in tumor regions predicted to be stiff. Our results define ILK as a key mechanotransducer in modulating breast CSC development in response to tissue mechanics and oxygen tension. Cancer Res; 76(18); 5277–87. ©2016 AACR.