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The prolyl isomerase cyclophilin A (CypA) regulates the Jak2/Stat5 pathway, which is necessary for mammary differentiation and the pathogenesis of breast cancer. In this study, we assessed the role of this isomerase during mammary gland development and erbB2-driven tumorigenesis. Genetic deletion of CypA resulted in delayed mammary gland morphogenesis and differentiation with corresponding decrease in Jak2/Stat5 activation; mammary gland cross-transplantation confirmed this defect was epithelial in nature. Analysis of mammary stem and progenitor populations revealed significant disruption of epithelial maturation. Loss of CypA in the erbB2 transgenic mouse model revealed a marked increase in mammary tumor latency that correlated with decreased Stat5 activation, associated gene expression, and reduced epithelial cell proliferation. These results demonstrate an important role for CypA in the regulation of Jak2/Stat5–mediated biology in mammary epithelium, identifying this isomerase as a novel target for therapeutic intervention. Significance: These findings reveal cyclophilin A functions in normal mammary epithelial development and ErbB2-driven mammary tumorigenesis and suggest therapies targeting cyclophilin A may be efficacious for breast cancer treatment. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/14/3877/F1.large.jpg. Cancer Res; 78(14); 3877–87. ©2018 AACR.

Estrogen receptor (ER) coregulator overexpression promotes carcinogenesis and/or progression of endocrine related-cancers in which steroid hormones are powerful mitogenic agents. Recent studies in our laboratory, as well as others, demonstrated that the estrogen receptor coregulator PELP1 is a proto-oncogene. PELP1 interactions with histone demethylase KDM1 play a critical role in its oncogenic functions and PELP1 is a prognostic indicator of decreased survival in patients with breast cancer. However, the in vivo significance of PELP1 deregulation during initiation and progression of breast cancer remains unknown. We generated an inducible, mammary gland-specific PELP1-expressing transgenic (Tg) mouse (MMTVrtTA-TetOPELP1). We found more proliferation, extensive side branching, and precocious differentiation in PELP1-overexpressing mammary glands than in control glands. Aged MMTVrtTA-TetOPELP1 Tg mice had hyperplasia and preneoplastic changes as early as 12 weeks, and ER-positive mammary tumors occurred at a latency of 14 to 16 months. Mechanistic studies revealed that PELP1 deregulation altered expression of a number of known ER target genes involved in cellular proliferation (cyclin D1, CDKs) and morphogenesis (EGFR, MMPs) and such changes facilitated altered mammary gland morphogenesis and tumor progression. Furthermore, PELP1 was hyper-phosphorylated at its CDK phosphorylation site, suggesting an autocrine loop involving the CDK–cyclin D1–PELP1 axis in promoting mammary tumorigenesis. Treatment of PELP1 Tg mice with a KDM1 inhibitor significantly reduced PELP1-driven hyperbranching, reversed alterations in cyclin D1 expression levels, and reduced CDK-driven PELP1 phosphorylation. These results further support the hypothesis that PELP1 deregulation has the potential to promote breast tumorigenesis in vivo and represent a novel model for future investigation into molecular mechanisms of PELP1-mediated tumorigenesis. Cancer Res; 74(24); 7395–405. ©2014 AACR.

The mammary gland is one of the few organs that continues to develop postnatally through stages including puberty, pregnancy, lactation, and involution. The gland is composed of epithelial and stromal cells that include fibroblasts, adipocytes, endothelial cells, nerve cells, and macrophages. Terminal end bud (TEB) structures are found exclusively in the pubertal developmental stage. The formation of TEBs and side branching drives mammary gland epithelial cell invasion into the mammary fat pad, continuing until the entire fat pad is filled. Pubertal mammary gland morphogenesis integrates a balance of epithelial cell proliferation, differentiation, and apoptosis. Several studies have shown that the interaction between mammary epithelial and stromal cells is crucial for the proper postnatal development of the mammary ductal tree. Interestingly, studies have shown that processes important in mammary gland development are often deregulated during breast cancer tumorigenesis. Thus, understanding the complex signaling network as well as the interactions between the different cell types during mammary gland development will be vital for elucidating the mechanisms underlying breast cancer progression and metastasis. Glycation is the non-enzymatic glycosylation of sugar moieties to biological macromolecules such as protein and DNA which produces reactive metabolites known as advanced glycation end products (AGE's). AGE content in the Western Diet has consistently increased over the last 50 years due to increased consumption of sugar laden and cheap processed/manufactured foods which are high in reactive AGE metabolites. AGE containing food can lead to the accumulation of AGEs in the body overtime leading to pro-inflammatory and pro-oxidant effects when signaling through receptor for advanced glycation end products (RAGE). Leading too many complications associated with diseases including diabetes, Alzheimer's, heart disease and cancer. Preliminary data in our lab has shown that AGEs also have an effect on phosphorylation and signaling of estrogen receptor α (ERα), a key receptor and signaling pathway in the regulation of mammary gland development during puberty. This observation, together with the links between diet, mammary gland development and immune cell recruitment lead us to examine the biological effects of a diet high in AGEs on pubertal mammary gland development in mice. We observed a significant disruption of normal pubertal mammary gland development in mice fed a high AGE diet when compared to mice fed a control diet. Mice fed the high AGE diet showed increases in TEB number as well as width, length and area. We also observed an increase in ductal branching and a decrease in ductal extension. Future studies will assess the role of macrophage recruitment to the developing gland, specifically around the TEBs based on its reported role in normal TEB function. We also plan to assess ERa signaling in mice fed the high AGE diet based on the reported role of estrogen signaling in ductal elongation. Citation Format: Krisanits BA, Nogueira LM, Findlay VJ, Turner DP. Diet, development and predisposition to breast cancer: The impact of sugar derived metabolites (AGEs) on pubertal mammary gland development [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-05-01.

Microtubule (MT) dynamics is essential to provide a correct apico-basal polarization of epithelial cells, by regulating the movement of vesicles and proteins within the cell and by ensuring a correct orientation of the mitotic spindle during cell division. Little is known about the role of the MT-dynamics and of MT-regulating proteins during development of the mammary gland, a dynamic organ that displays a complex and finely organized apico-basal architecture. Stathmin, a MT-destabilizing protein, is often overexpressed in breast cancer (BC). By studying knockout (STM KO) mice we observed that STM KO females were unable to nurse their offspring. This phenotype was accompanied by defective development of the mammary gland, particularly evident during pregnancy and post-partum phases. Our results showed that stathmin absence mainly impinged on two key features of the mammary epithelial organization, polarity and proliferation. In vitro, normal mammary epithelial cells silenced for stathmin were not able to form organized acini in 3D-matrices. In vivo, when stathmin was KO, many molecules involved in mammary gland functions were not properly localized, indicating a disruption of the apico-basal polarity. Furthermore, loss of stathmin strongly decreased proliferation and induced disoriented positioning of the mitotic spindle in dividing epithelial cells, preventing the correct orientation of the daughter cells. Loss of polarity and deregulated proliferation are tightly linked to tumorigenesis. We therefore examined the role of stathmin also in the context of HER2-driven tumorigenesis. To this end, we used a transgenic mouse model expressing a constitutively active form of HER2 (Δ16HER2), which is an alternatively spliced, very aggressive form of HER2, and intercrossed them with STM WT and KO mice. We analyzed the role of stathmin in both early and late stages of tumorigenesis. STM KO mice displayed a significantly reduced number of pre-neoplastic foci and this phenotype was maintained also in late stages of tumorigenesis. In WT mice, pre-neoplastic lesions expressed high level of stathmin compared with normal tissue supporting the idea that stathmin played an active role in tumor initiation. Analysis of proliferation in tumors revealed that loss of stathmin strongly decreased the mitotic rate of Δ16HER2-transformed cells. Furthermore, we assessed that stathmin plays a cell autonomous function in tumor initiation. Our study has shed new light in the field of mammary gland development and of breast cancer biology. Collectively, our data indicate that loss of stathmin by altering the dynamics of microtubules, causes a profound disorganization of the normal mammary gland architecture and a delay in initiation of the tumorigenic process. We have uncovered new functions of stathmin that could have important implications in BC, also in consideration that several microtubules acting drugs are currently employed in the therapy of BC patients Citation Format: Ilenia Segatto, Mara De Marco Zompit, Gian Luca Rampioni Vinciguerra, Francesca Citron, Sara D'Andrea, Tiziana Perin, Gustavo Baldassarre, Barbara Belletti. Stathmin regulates mammary gland morphogenesis and tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1460.

The tumor suppressor BRCA1 interacts with many proteins and undergoes multiple modifications on DNA damage. ATM, a key molecule of the DNA damage response, phosphorylates S1189 of BRCA1 after γ-irradiation. S1189 of BRCA1 is known as a unique ATM phosphorylation site in BRCA1 exon 11. To study the functions of ATM-dependent phosphorylation of BRCA1-S1189, we generated a mouse model carrying a mutation of S1152A (S1152 in mouse Brca1 corresponds to S1189 in human BRCA1) by gene targeting. Brca1S1152A/S1152A mice were born at the expected ratio, unlike that seen in previous studies of Brca1-null mice. However, 36% of Brca1S1152A/S1152A mice exhibited aging-like phenotypes including growth retardation, skin abnormalities, and delay of the mammary gland morphogenesis, with an increase in apoptosis. Mutant mice were hypersensitive to high doses of γ-irradiation, displaying shortened life span and reduction in intestinal villus size, associated with increased apoptosis. Aging-unaffected 18-month-old Brca1S1152A/S1152A female mice also showed mammary gland abnormalities with increased levels of cyclin D1 and phospho-ER-α, such as Brca1-Δ11 mutation. On low-dose γ-irradiation, they suffered a marked increase in tumor formation with an abnormal coat pattern. Furthermore, Brca1S1152A/S1152A embryonic fibroblasts failed to accumulate p53 on γ-irradiation with delayed phosphorylation of p53-S23. These observations indicate that ATM-mediated phosphorylation of S1189 is required for BRCA1 functions in the modulation of DNA damage response and in the suppression of tumor formation by regulating p53 and apoptosis. [Cancer Res 2009;69(24):9291–300]

Epimorphin/syntaxin-2 (EPM) is a plasma membrane–anchored protein that has at least two distinct functions depending on its membrane topology: vesicle fusion when localized to the cytoplasmic surface and morphogenic signaling when localized to the extracellular surface. Transgenic mice that express full-length extracellular EPM fused to the NH2-terminal signal sequence of interleukin-2, under the control of the whey acidic protein (WAP) gene promoter, exhibit aberrant mammary gland morphogenesis associated with increased expression of CCAAT enhancer binding protein β (C/EBPβ). Here we report that aged nulliparous and uniparous female WAP-EPM transgenic mice develop alveolar hyperplasias and well-differentiated adenocarcinomas that express high levels of C/EBPβ, keratin-14, matrix metalloproteinase-3, and β-catenin. This study reveals another pathway in which overexpression and alteration of a normal morphogenic process promote the development of cancer in the mammary gland.

It has long been appreciated that the processes of tissue morphogenesis and tumor initiation/progression share many common features. This is particularly the case for signaling networks involved in cell proliferation, survival, and motility. The focus of the current study is on a signaling complex comprised of the adaptor molecules p130Cas (Cas) and Breast Cancer Antiestrogen Resistance 3 (BCAR3), which together are potent activators of the non-receptor tyrosine kinase c-Src. BCAR3 and Cas are present in a stable molecular complex in breast tumor cells where they have been shown to play a role in the regulation of cell adhesion and motility, both of which are obligate elements of tumor cell proliferation and invasion. In this study, we sought to determine whether the biological activities attributed to BCAR3/Cas in breast cancer cells were dependent upon their interaction, and whether these molecules also might function during breast morphogenesis. We show that the entire pool of BCAR3 is in complex with Cas in invasive breast tumor cells and that these proteins co-localize in dynamic cellular adhesions. While accumulation of BCAR3 in adhesions does not require Cas binding, a direct interaction between BCAR3 and Cas is necessary for efficient dissociation of BCAR3 from adhesions. The dissociation rates of Cas and two other adhesion proteins, α-actinin and talin, are also significantly slower in the presence of a Cas-binding mutant of BCAR3, suggesting that turnover of the entire adhesion complex is delayed under these conditions. As is the case for adhesion turnover, BCAR3-Cas interactions were found to be important for BCAR3-mediated breast tumor cell invasion in Matrigel. Previous work showed that BCAR3 is a potent activator of Rac1, which in turn is an important regulator of adhesion dynamics and invasion. We show that, as is the case for adhesion turnover, Rac1 activation by BCAR3 also requires its interaction with Cas. Together, these data show that the ability of BCAR3 to promote adhesion disassembly, tumor cell invasion, and Rac1 activity, are all dependent on its ability to bind to Cas, thus implicating the BCAR3/Cas complex as an important functional unit in invasive breast tumor cells. To determine whether this was also the case in breast tumors, we set out to measure BCAR3 protein expression in tumor samples. We reasoned that BCAR3 expression could potentially serve as a surrogate marker for the activity of the BCAR3/Cas complex since all of the BCAR3 in breast tumor cells is associated with Cas. Preliminary data indicate that BCAR3 protein levels are extremely low in normal mammary epithelial cells but that its expression is elevated in multiple breast tumor subtypes. BCAR3 is also expressed during tumor progression in the MMTV-polyoma virus middle T murine breast tumor model. Studies are now being performed in the mouse to determine whether BCAR3 expression in breast tumors promotes tumor progression and metastasis. If this is the case, then targeting the BCAR3/Cas complex and its downstream effectors could be a viable approach for treating invasive breast cancers. Like many signaling pathways that are involved in cancer development, we hypothesize that the BCAR3/Cas signaling complex also plays a role in normal morphogenesis. Analysis of BCAR3 protein in developing mammary glands showed that BCAR3 expression is highest during puberty and is downregulated in fully developed glands. We are currently modeling mammary gland morphogenesis using organoid cultures to investigate the potential role of BCAR3/Cas signaling in this process. Determining how signaling through BCAR3/Cas supports mammary gland development will contribute to our understanding of how elevated signaling through this complex may promote breast cancer progression. Citation Format: Allison M. Cross, Ashley L. Wilson, Michael S. Guerrero, Keena S. Thomas, Alexia I. Bachir, Ryan A. Llewellyn, Kristopher E. Kubow, A Rick Horwitz, Amy H. Bouton. A role for the BCAR3/Cas signaling complex in breast tumor progression, metastasis and normal breast morphogenesis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B17.

Spy1A is a unique cell cycle activator known to mediate cell cycle progression and override the DNA damage response. This study focused on determining the role of this protein on postnatal mammary gland morphogenesis and neoplasia. Herein, we show that Spy1A levels are tightly regulated during mammary gland development and that ectopic expression stimulates precocious development and results in disrupted morphology of the gland. This follows the same trend as the oncogene c-Myc, and we show that Spy1A expression is regulated downstream of c-Myc signaling. Importantly, we show that overexpression of Spy1A accelerates tumorigenesis in vivo. Collectively, this work is the first report that the Spy1/RINGO family of proteins may play an essential role in regulating both normal and abnormal growth processes in the breast. [Cancer Res 2008;68(10):3591–600]

The Hedgehog (Hh) signaling pathway functions as an organizer in embryonic development. Genetic analysis has demonstrated a critical role for the Hh pathway in mammary gland morphogenesis. Disruption of Patched1, a component of the Hh pathway, results in abnormal growth of mammary duct. Recent studies have shown constitutive activation of the Hh pathway in various types of malignancies. However, it remains unclear whether this pathway is activated in human breast cancer. Here, we determined the expression of the components, including Sonic Hh, Patched1, and Gli1, of the Hh pathway by immunohistochemical staining in a series of 52 human breast carcinomas. All of 52 tumors display staining of high intensity for Gli1 when compared with adjacent normal tissue. The nuclear staining ratio of Gli1 correlates with expression of estrogen receptor and histologic type. Exposure to cyclopamine, a steroidal alkaloid that blocks the Hh pathway, suppresses expression of Gli1 and the growth of the Hh pathway-activated breast carcinoma cells. These data indicate that the Hh pathway is a new candidate for therapeutic target of breast cancer.

While integrin αvβ3 expression is linked to breast cancer progression its role in epithelial development is unclear. Here, we show that αvβ3 plays a critical role in adult mammary stem cells (MaSCs) during pregnancy. While αvβ3 is a luminal progenitor marker in the virgin gland, we noted increased αvβ3 expression in MaSCs at mid-pregnancy. Accordingly, mice lacking αvβ3 or expressing a signaling deficient receptor showed defective mammary gland morphogenesis during pregnancy. This was associated with decreased MaSC expansion, clonogenicity and expression of Slug, a master regulator of MaSCs. Surprisingly, αvβ3 deficient mice displayed normal development of the virgin gland with no effect on luminal progenitors. TGFβ2, but not RANKL, induced αvβ3 leading to Slug nuclear accumulation and MaSC clonogenicity. In human breast cancer cells, αvβ3 was necessary and sufficient for Slug activation and tumorsphere formation. Thus, pregnancy-associated MaSCs require a TGFβ2/αvβ3/Slug pathway, which may also contribute to breast cancer progression. Citation Format: Jay S. Desgrosellier, Jacqueline Lesperance, Laetitia Seguin, Sanford J. Shattil, David A. Cheresh. Integrin αvβ3 drives Slug activation and stemness in the pregnant and neoplastic mammary gland. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3034. doi:10.1158/1538-7445.AM2014-3034

Transforming growth factor β (TGFβ) is a potent and context dependent regulator of tumor progression. TGFβ promotes the lung metastasis of basal-like (but not the luminal-like) breast cancer. Here, we demonstrated that fascin, a pro-metastasis actin bundling protein, was a direct target of the canonical TGFβ-Smad4 signaling pathway in basal-like breast cancer cells. TGFβ and Smad4 induced fascin overexpression by directly binding to a Smad binding element on the fascin promoter. Through data mining, we identified GATA3, a transcription factor crucial for mammary gland morphogenesis and luminal differentiation, as a potential regulator of fascin overexpression. When ectopically expressed in basal-like breast cancer cells, GATA-3 abrogated Smad4-mediated overexpression of fascin and other TGFβ response genes, invadopodium formation, cell migration and invasion, suggesting suppression of the canonical TGFβ-Smad4 signaling axis. Mechanistically, GATA3 might abrogate TGFβ and Smad4-mediated fascin overexpression by abolishing the interactions between Smad4 and its DNA binding elements, potentially through physical interactions between GATA3 and Smad3/4. Our findings provide mechanistic insight into how TGFβ-mediated cell motility and invasiveness are differentially regulated in breast cancer. Citation Format: Jianwei Sun, Huifang He, Smitha Pillai, Srikumar Chellappan, Shengyu Yang. GATA3 abrogates TGFbeta-mediated breast cancer invasion and metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5001. doi:10.1158/1538-7445.AM2014-5001

Our aim is to better understand the mechanisms dictating the early dissemination of cells from pre-malignant lesions, as well as those controlling cell fate decisions at secondary sites to develop more effective therapies for eradicating these populations. The majority of breast cancer patients die from metastatic disease, and not from their primary tumor. The current paradigm argues that dissemination of tumor cells can only occur from invasive lesions observed during late stages of the disease. However, clinical evidence suggests that “pre-malignant” mammary epithelial cells can disseminate and accumulate in target organs like the bone. This can occur from non-invasive lesions such as those found in Ductal Carcinoma in Situ (DCIS) patients. Thus, “early” disseminated tumor cells (DTCs) might be critical contributors to metastasis, but the mechanisms behind these events remain largely unknown. We had shown that p38-alpha/beta signaling maintains mammary gland ductal architecture by promoting anoikis and preventing disorganization of the luminal and basal epithelial compartments. We propose that, in addition to regulating mammary gland morphogenesis, p38-alpha/beta regulates a switch controlling early dissemination. In clinical samples we have found that ErbB2+ DCIS or invasive lesions show a down-regulation of the p38-signaling pathway, suggesting a role for the inhibition of p38-alpha/beta during cancer progression. In MMTV-Neu mammary glands ErbB2 is inversely correlated to membranous beta-catenin (inactive beta-catenin). Further, loss of P-ATF2 (p38 target) also coincides with reduced E-cadherin in this ErbB2+ epithelium. We also reveal that inhibition of p38-alpha/beta in MCF10A-ErbB2 or “pre-malignant” MMTV-ErbB2 acini results in increased invasion and disruption of normal acinar structure. In the MCF10A-ErbB2 model this correlated with E-cadherin down-regulation and induction of an 11-gene Wnt signaling EMT signature (e.g. up-regulation of Wnt5a/b, Wnt11, SNAIL, TWIST, N-cadherin, ZEB2, and SLUG). This EMT signature was also induced simply by p38-alpha/beta inhibition or ErbB2 overexpression in parental MCF10A cells. Further, treatment of MCF10A cells with Wnt3a inhibited p38-alpha and ATF2 phosphorylation. Inhibition of p38-alpha/beta signaling in MMTV-ErbB2 mice strongly increased the accumulation of pre-malignant MECs (detected by surface ErbB2) in the lungs and bone marrow. However, at least in lungs, >80% of these DTCs remained dormant as measure by P-Rb staining. We propose that in pre-malignant lesions p38-alpha/beta acts as an inhibitor to ErbB2-induced EMT, anoikis resistance and early dissemination. We further propose that Wnt signaling executes the EMT and also the silencing of p38-alpha/beta signaling to allow for early dissemination. This abstract is also presented as Poster B74. Citation Format: Kathryn Harper, Maria Soledad Sosa, Alvaro Avivar-Valderas, Huei-Chi Wen, Julio Aguirre-Ghiso. ErbB2-induced epithelium to mesenchyme transition (EMT) is blocked by p38α/β signaling to restrict early dissemination in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr PR14.

Profilin1 (Pfn1), an essential control element for actin polymerization, plays indispensable role in migration and proliferation of normal cells. Seemingly contrary to these essential cellular functions of Pfn1, its expression is downregulated in breast cancer, the significance of which is unclear. In this study, expression profiling of Pfn1 in human breast cancer specimens of various histopathological stages correlate lower Pfn1 expression levels with propensity to metastasize. Xenograft studies uncover a unique dichotomous role of Pfn1 in early vs late steps of breast cancer metastasis. Specifically, loss of Pfn1 expression promotes dissemination of breast cancer cells from the primary mammary tumor but metastatic colonization of breast cancer cells is critically dependent on Pfn1's function. In the context of dissemination, breast cancer cells exhibit increased sustenance of PI3K signaling and acquire a hyper-invasive phenotype (marked by MMP9 upregulation, faster invasion through collagen matrix) involving a PI3K-PI(3,4)P2 signaling axis when Pfn1 expression is suppressed. In addition, Pfn1-deficient breast cancer cells display increased proficiency to cross through an endothelial cell barrier (an obligatory step for vascular dissemination) in a VEGF-dependent manner, respectively. Gene expression profiling combined with biochemical analyses of secreted proteins show that loss of Pfn1 impacts expression of several other pro-invasive and pro-metastatic genes. Finally, correlated with hyper-invasive phenotype of breast cancer cells caused by Pfn1 depletion, we found that mammary-specific deletion of Pfn1 gene expedites ductal outgrowth. In summary, contrasting the well-established pro-migratory role of Pfn1 in the physiological contexts, our findings set the first in vivo example of a context-specific effect of loss of Pfn1 in cell motility-dependent processes such as mammary gland morphogenesis and metastatic dissemination. Citation Format: Partha Roy. Dichotomus role of profilin in early vs. late steps of breast cancer metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B42.

Breast tissue development and homeostasis are tightly regulated by mechanical cues. Alterations in tensional homeostasis characterize breast cancer. The challenge is to clarify how chemical and mechanical cue collaborate to regulate breast tissue function and to determine when and how these forces regulate breast cancer behavior. The Weaver group has been studying how cells in the breast sense and transduce mechanical cues to regulate their behavior and how altered mechanical force compromises breast tissue homeostasis to drive cancer (Dufort et al., Nature Mol Biol Rev 2011). Our work focuses on clarifying the role of force in normal breast behavior and in breast cancer initiation, transformation, metastasis and treatment response. We showed that extracellular matrix (ECM) stiffness, which is tightly controlled through matrix concentration, topology and posttranslational modifications, is a critical isometric force that modulates mammary gland morphogenesis and tumorigenesis. We demonstrated that the ECM associated with the mammary epithelium progressively stiffens as the tissue transforms and that breast cancer subtype is characterized by increasing ECM stiffness. Thus, normal human breast tissue is quite soft, the ECM associated with fibrocystic lesions is twice as stiff, ER/PR+ve invasive breast cancers are 4 fold stiffer and triple negative breast lesions are the stiffest (6-10 times stiffer). We showed that a stiffened ECM modifies breast tissue behavior by promoting the assembly of integrin focal adhesions which potentiate growth factor receptor signaling and induce cytoskeletal remodeling and actomyosin contractility (Paszek et al., Cancer Cell 2005; Paszek et al., PLOS Computational 2009). We determined that ECM stiffness also potentiates the tumor promoting effect of oncogenes such as Ras, EGFR and ErbB2 by increasing the activity of ERK, wnt and PI3 kinase and inducing tissue fibrosis. Inducing ECM stiffness promotes the malignant transformation of pre malignant oncogenically-primed mammary epithelial cells, whereas inhibiting ECM stiffening prevents breast tissue fibrosis and tumor progression and metastasis (Levental et al., CELL 2009). Importantly, the tissues response to force also depends upon the cellular mechanoresponsive machinery and we determined that oncogenic transformation and elevated chemokine and cytokine signaling induces aberrant force sensing and elevated breast tumor tension. Most notably we demonstrated that oncogenic signaling through Ras signaling to ERK induces ECM remodeling and stiffening which are required to stimulate wnt thereby driving tumor cell invasion to induce an epithelial to mesenchymal like transition that potentiates breast tumor aggression and metastasis. More recently we found that ECM stiffness dictates the efficacy of treatment response - with high ECM stiffness potentiating chemo, immune and radiation-induced tumor regression and apoptosis. The clinical relevance of these findings are now being investigated with collaborators at UCSF and Duke. (supported by DOD BCRP W81XWH-05-1-0330 and NCI U54CA143836-01, R01 CA138818-01A1, U01 CA151925-01, RO1 CA085492, and R01 CA140663-01A2 to VMW, NIH grant 1U01ES009458-01 to ZW and NCI SPORE P50 CA058207 to VMW, CP, & SH). Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr BS3-1.