Irene Acerbi

Tissue mechanics modulate microRNA-dependent PTEN expression to regulate malignant progression.

Tissue mechanics regulate development and homeostasis and are consistently modified in tumor progression. Nevertheless, the fundamental molecular mechanisms through which altered mechanics regulate tissue behavior and the clinical relevance of these changes remain unclear. We demonstrate that increased matrix stiffness modulates microRNA expression to drive tumor progression through integrin activation of β-catenin and MYC. Specifically, in human and mouse tissue, increased matrix stiffness induced miR-18a to reduce levels of the tumor suppressor phosphatase and tensin homolog (PTEN), both directly and indirectly by decreasing levels of homeobox A9 (HOXA9). Clinically, extracellular matrix stiffness correlated directly and significantly with miR-18a expression in human breast tumor biopsies. miR-18a expression was highest in basal-like breast cancers in which PTEN and HOXA9 levels were lowest, and high miR-18a expression predicted poor prognosis in patients with luminal breast cancers. Our findings identify a mechanically regulated microRNA circuit that can promote malignancy and suggest potential prognostic roles for HOXA9 and miR-18a levels in stratifying patients with luminal breast cancers.

Stromally Derived Lysyl Oxidase Promotes Metastasis of Transforming Growth Factor-β–Deficient Mouse Mammary Carcinomas

The tumor stromal environment can dictate many aspects of tumor progression. A complete understanding of factors driving stromal activation and their role in tumor metastasis is critical to furthering research with the goal of therapeutic intervention. Polyoma middle T-induced mammary carcinomas lacking the type II TGF-β receptor (PyMT(mgko)) are highly metastatic compared with control PyMT-induced carcinomas (PyMT(fl/fl)). We hypothesized that the PyMT(mgko)-activated stroma interacts with carcinoma cells to promote invasion and metastasis. We show that the extracellular matrix associated with PyMT(mgko) tumors is stiffer and has more fibrillar collagen and increased expression of the collagen crosslinking enzyme lysyl oxidase (LOX) compared with PyMT(fl/fl) mammary carcinomas. Inhibition of LOX activity in PyMT(mgko) mice had no effect on tumor latency and size, but significantly decreased tumor metastasis through inhibition of tumor cell intravasation. This phenotype was associated with a decrease in keratin 14-positive myoepithelial cells in PyMT(mgko) tumors following LOX inhibition as well as a decrease in focal adhesion formation. Interestingly, the primary source of LOX was found to be activated fibroblasts. LOX expression in these fibroblasts can be driven by myeloid cell-derived TGF-β, which is significantly linked to human breast cancer. Overall, stromal expansion in PyMT(mgko) tumors is likely caused through the modulation of immune cell infiltrates to promote fibroblast activation. This feeds back to the epithelium to promote metastasis by modulating phenotypic characteristics of basal cells. Our data indicate that epithelial induction of microenvironmental changes can play a significant role in tumorigenesis and attenuating these changes can inhibit metastasis. Cancer Res; 73(17); 5336-46. ©2013 AACR.

Collagen architecture in pregnancy-induced protection from breast cancer

Summary The reduction in breast cancer risk attributed to early-age pregnancy is mediated in part by changes in the mammary epithelium. Here, we address the role of the mammary stroma in this protection. Utilizing tumor cells capable of transitioning from indolent to proliferative or invasive states, we demonstrate that mammary extracellular matrix (ECM) from parous rats (parous matrix) decreases tumor growth and impedes cellular phenotypes associated with tumor cell invasion compared with that observed using nulliparous matrix. Proteomic analysis identifies an increased abundance of collagen I in parous matrix, an observation extended to breast tissue of parous women. Given the pro-tumorigenic attributes of fibrillar collagen, these results were unexpected. Second-harmonic generation imaging and atomic force microscopy revealed that the abundant collagen observed in the mammary glands of parous rats is less linearized and associated with a decrease in stromal stiffness, implicating collagen organization and stiffness in parity-induced protection. Using 3D cell culture models, we demonstrate that linearized (fibrillar) collagen I induces cellular phenotypes consistent with an invasive behavior in mammary tumor cells and alters the subcellular distribution of &bgr;1 integrin. Conversely, high-density non-fibrillar collagen I induces tumor-suppressive attributes, including increases in junctional E-cadherin in tumor cells, upregulation of genes encoding components of cell–cell junctions, and downregulation of mesenchymal-specific and metalloproteinase-encoding genes. These data show that collagen organization, rather than density alone, is a key contributor to the invasive phenotype. Furthermore, our data show that parity alters the composition and organization of mammary ECM, particularly fibrillar collagen, in a manner consistent with tumor suppression.

Integrin-Specific Mechanoresponses to Compression and Extension Probed by Cylindrical Flat-Ended AFM Tips in Lung Cells

Cells from lung and other tissues are subjected to forces of opposing directions that are largely transmitted through integrin-mediated adhesions. How cells respond to force bidirectionality remains ill defined. To address this question, we nanofabricated flat-ended cylindrical Atomic Force Microscopy (AFM) tips with ∼1 µm2 cross-section area. Tips were uncoated or coated with either integrin-specific (RGD) or non-specific (RGE/BSA) molecules, brought into contact with lung epithelial cells or fibroblasts for 30 s to form focal adhesion precursors, and used to probe cell resistance to deformation in compression and extension. We found that cell resistance to compression was globally higher than to extension regardless of the tip coating. In contrast, both tip-cell adhesion strength and resistance to compression and extension were the highest when probed at integrin-specific adhesions. These integrin-specific mechanoresponses required an intact actin cytoskeleton, and were dependent on tyrosine phosphatases and Ca2+ signaling. Cell asymmetric mechanoresponse to compression and extension remained after 5 minutes of tip-cell adhesion, revealing that asymmetric resistance to force directionality is an intrinsic property of lung cells, as in most soft tissues. Our findings provide new insights on how lung cells probe the mechanochemical properties of the microenvironment, an important process for migration, repair and tissue homeostasis.

Respiratory impedance during weaning from mechanical ventilation in a mixed population of critically ill patients

BACKGROUND Worsening of respiratory mechanics during a spontaneous breathing trial (SBT) has been traditionally associated with weaning failure, although this finding is based on studies with chronic obstructive pulmonary disease patients only. The aim of our study was to assess the course of respiratory impedance non-invasively measured by forced oscillation technique (FOT) during a successful and failed SBT in a mixed population. METHODS Thirty-four weaning trials were reported in 29 consecutive mechanically ventilated patients with different causes of initiation of ventilation. During the SBT, the patient was breathing through a conventional T-piece connected to the tracheal tube. FOT (5 Hz, +/- 1 cm H(2)O, 30 s) was applied at 5, 10, 15, 20, 25, and 30 min. Respiratory resistance (Rrs) and reactance (Xrs) were computed from pressure and flow measurements. The frequency to tidal volume ratio f/V(t) was obtained from the flow signal. At the end of the trial, patients were divided into two groups: SBT success and failure. RESULTS Mixed model analysis showed no significant differences in Rrs and Xrs over the course of the SBT, or between the success (n=16) and the failure (n=18) groups. In contrast, f/V(t) was significantly (P<0.001) higher in the failure group. CONCLUSIONS Worsening of respiratory impedance measured by FOT is not a common finding during a failed SBT in a typically heterogeneous intensive care unit population of mechanically ventilated patients.

Abstract OT3-03-01: Preference-Tolerant randomized trial of risk-based vs. annual breast cancer screening: WISDOM study in progress

Purpose: Women Informed to Screen Depending on Measures of risk (WISDOM) trial is a pragmatic study comparing two real world approaches to clinical care for breast screening: annual screening versus personalized screening. The novelty of the personalized arm of the study is that we are combining known risk factors (age, family history, history of breast disease, ethnicity, BIRADS breast density, and genetics) into a single risk assessment model. All components of the model have been tested and established, but have never been used jointly. The goal of the WISDOM study is to examine the effectiveness of personalized breast cancer screening and to bring objective recommendations to the current mammography screening debate. Methods: The WISDOM trial will enroll 100,000 women with a preference-tolerant design that will determine if risk-based screening vs. annual screening, is as safe, less morbid, enables prevention, and is preferred by women. Women 40 - 74 years of age with no history of breast cancer or DCIS, and no previous double mastectomy can join the study from the WISDOM Study website (wisdomstudy.org). All participants sign up, elect randomization or self-select the study arm, provide electronic consent using DocuSign (eConsent), and sign a Medical Release Form. For all participants, 5-year risk of developing breast cancer is calculated according to the Breast Cancer Screening Consortium (BCSC) model. For participants in the personalized arm, the overall 5-year risk BCSC score is combined with a Polygenic Risk Score, based on a genetic test including mutations in 9 genes (BRCA1, BRCA2, TP53, PTEN, STK11, CDH1, ATM, PALB2, and CHEK2) and a panel of 75 common single nucleotide polymorphisms known to increase breast cancer risk. Risk stratification will determine frequency of screening. The study is registered on ClinicalTrials.gov as NCT02620852. Results: As of June 12th 2017, the WISDOM study is live at all UC medical centers and recruitment is open to all eligible women in California. Up to date 4,769 eligible women registered at all sites. 2,823 women have consented in the trial. 64% were randomized and 36% chose their screening arm. A pilot was conducted to test the logistics of online participation and examine the acceptance of the study design and approach. We are partnering with health insurance companies and self-insured companies to reach our recruitment goal. Conclusions: Enrollment will be completed by end of 2018. Acknowledgment: support by the Patient-Centered Outcomes Research Institute (PCORI), PCS-1402-10749 to L.J.E. (*) Authors equally contributed to this work. Citation Format: Acerbi I, Abihider K, Ling J, Layton T, DeRosa D, Madlensky L, Tice J, Shieh Y, Ziv E, Sarrafan S, Firouzian R, Tong B, Blanco A, Lee V, Heditsian D, Brain S, Kaplan C, Borowsky A, Anton-Culver H, Naeim A, Cink T, Stover Fiscalini A, Parker B, van 9t Veer L, Wisdom Study and Athena Breast Health Network Investigators and Advocate Partners, LaCroix A, Esserman L. Preference-Tolerant randomized trial of risk-based vs. annual breast cancer screening: WISDOM study in progress [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr OT3-03-01.

Abstract C24: Interplay between ECM stiffness, miR-203 and mammographic density

High mammographic density (MD) is associated with a greater risk of breast cancer and is characterized by high extracellular matrix (ECM) collagen. Elevated collagen increases ECM stiffness and can promote the malignant transformation of oncogenically-primed mammary epithelial cells in culture and in vivo. Whether or not high MD reflects a breast stroma that is stiff and if ECM stiffness accounts for the elevated breast cancer risk in high MD tissue and how has yet to be resolved. To address this question we are conducting a comprehensive biophysical and molecular analysis of BIRADS status in prophylactic mastectomies in women with low (BIRADS 1) versus high MD (BIRADS 4). Data revealed that the intra-lobular ECM associated with the terminal end-buds contains anisotropic compliant and relaxed collagen fibrils. By contrast, the inter-lobular ECM contains stiff, oriented collagen fibrils. Preliminary data suggest that the ECM associated with the terminal end-buds in the upper outer quadrant may be stiffer in women with high MD as compared to low MD. These same tissues also expressed lower levels of the microRNA miR-203, which has been implicated in epithelial-to-mesenchymal transition (EMT) inhibition. Consistently, in vitro studies revealed that miR-203 was repressed in MECs cultured on a stiff ECM. We also found that a stiffened mammary ECM decreases miR-203 expression and promotes an EMT. Additionally, inhibiting ECM stiffening restored miR-203 levels and reduced an EMT. Moreover and importantly, we showed that triple negative breast tumors that often express EMT markers have reduced miR-203 levels. Studies are underway to elaborate these preliminary findings and to assess their clinical relevance. (This work is supported by the: NIH NSRA T32 CA 108462-11 to Ivory Dean, Susan G Komen PDF12230246 to Irene Acerbi, W81XWH-13-1-0216, USMRAA DOD-BCRP BC122990, NIH R01 CA138818-01A1 and NIH R01 CA192914-01 to Valerie Weaver.) Citation Format: Ivory Dean, Irene Acerbi, Janna Mouw, Quanming Shi, Yunn-Yi Chen, Jan Liphardt, Shelley Hwang, Valerie Weaver. Interplay between ECM stiffness, miR-203 and mammographic density. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr C24.

Abstract A098: A role for fibrosis in promoting pro-tumor immune response in breast cancer

We established a positive correlation between a fibrotic phenotype in human breast tumors — especially the Her2 and basal-like breast cancer subtypes — and CD45 and CD68 positive immune cell infiltration. We were interested in elucidating how this fibrotic phenotype may influence the immune response. To address this question, we examined if matrix stiffness alters the function of STAT3, a central regulator of tumor inflammation. We hypothesize that tissue fibrosis promotes STAT3 signaling in mammary tumor cells and alter the cytokine milieu to induce a pro-tumor immune response. We found that ECM stiffness directly enhanced STAT3 phosphorylation in tumor cells both in vitro and in vivo. Our data suggest the fibrotic phenotype promotes STAT3 activity, enhancement of which may drive a pro-tumor immune response. Indeed, we observed several alterations in cytokines and immune cell populations upon STAT3 ablation consistent with anti-tumor immune response. Interestingly, our data also suggest STAT3 knockout in tumor cells doesn9t necessary influence immune cell infiltration, but rather their differentiation in mammary tumors. Finally, we investigated if matrix stiffness has potentiated macrophage differentiation when cultured with specific immunosuppressive cytokines. Overall, our work reveals a novel mechanistic insight into how a pro-tumor immune response stems from the interplay between fibrosis and STAT3 signaling in tumor cells. As such, our findings may stimulate an interest in exploring combinational treatment options with anti-fibrotic agents and immunotherapy. Citation Format: Ori Maller, Luke Cassereau, Allison Drain, Brian Ruffell, Irene Acerbi, Miranda Broz, Jennifer Munson, Melody Swartz, Matthew Krummel, Lisa Coussens, Valerie Weaver. A role for fibrosis in promoting pro-tumor immune response in breast cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A098.

Abstract 5100: Interplay between ECM stiffness, miR-203 and mammographic density

High mammographic density (MD) is associated with a greater risk of breast cancer and is characterized by high extracellular matrix (ECM) collagen. Elevated collagen increases ECM stiffness and can promote the malignant transformation of oncogenically-primed mammary epithelial cells in culture and in vivo. Whether or not high MD reflects a breast stroma that is stiff and if ECM stiffness accounts for the elevated breast cancer risk in high MD tissue has yet to be resolved. To address this question we are conducting a comprehensive biophysical and molecular analysis of BIRADS status in prophylactic mastectomies in women with low (BIRADS 1) versus high MD (BIRADS 4). Data revealed that the intra-lobular ECM associated with the terminal end-buds contains anisotropic compliant and relaxed collagen fibrils. By contrast, the inter-lobular ECM contains stiff, oriented collagen fibrils. Preliminary data suggest that the ECM associated with the terminal end-buds in the upper outer quadrant may be stiffer in women with high MD as compared to low MD. These same tissues also expressed lower levels of the microRNA miR-203, which has been implicated in epithelial-to-mesenchymal transition (EMT) inhibition. Consistently, in vitro studies revealed that miR-203 was repressed in MECs cultured on a stiff ECM. We also found that a stiffened mammary ECM decreased miR-203 expression and promoted an EMT. Additionally, inhibiting ECM stiffening restored miR-203 levels and reduced an EMT. Moreover and importantly, we showed that triple negative breast tumors that often express EMT markers have reduced miR-203 levels. Studies are underway to elaborate these preliminary findings and to assess their clinical relevance. (This work is supported by the: NIH NSRA T32 CA 108462-11 to Ivory Dean, Susan G Komen PDF12230246 to Irene Acerbi, W81XWH-13-1-0216, USMRAA DOD-BCRP BC122990, NIH R01 CA138818-01A1 and NIH R01 CA192914-01 to Valerie Weaver.) Citation Format: Ivory Dean, Irene Acerbi, Alfred Au, Yunn-Yi Chen, Shelley Hwang, Valerie Weaver. Interplay between ECM stiffness, miR-203 and mammographic density. [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 5100.

Extracellular matrix stiffness modulates tissue inflammation to promote breast tumor aggression

Author(s): Acerbi, I; Hwang, S; Munson, J; Au, A; Zheng, S; Yu, H; Mouw, J; Lakins, J; Swartz, M; Shi, Q; Liphardt, J; Ruffell, B; Coussen, LM; Yunn-Yi, C; Weaver, VM