MMTV-PyMT Breast Cancer Model Research Articles

PTHrP induces STAT5 activation, secretory differentiation and accelerates mammary tumor development

BackgroundParathyroid hormone-related protein (PTHrP) is required for embryonic breast development and has important functions during lactation, when it is produced by alveolar epithelial cells and secreted into the maternal circulation to mobilize skeletal calcium used for milk production. PTHrP is also produced by breast cancers, and GWAS studies suggest that it influences breast cancer risk. However, the exact functions of PTHrP in breast cancer biology remain unsettled.MethodsWe developed a tetracycline-regulated, MMTV (mouse mammary tumor virus)-driven model of PTHrP overexpression in mammary epithelial cells (Tet-PTHrP mice) and bred these mice with the MMTV-PyMT (polyoma middle tumor-antigen) breast cancer model to analyze the impact of PTHrP overexpression on normal mammary gland biology and in breast cancer progression.ResultsOverexpression of PTHrP in luminal epithelial cells caused alveolar hyperplasia and secretory differentiation of the mammary epithelium with milk production. This was accompanied by activation of Stat5 and increased expression of E74-like factor-5 (Elf5) as well as a delay in post-lactation involution. In MMTV-PyMT mice, overexpression of PTHrP (Tet-PTHrP;PyMT mice) shortened tumor latency and accelerated tumor growth, ultimately reducing overall survival. Tumors overproducing PTHrP also displayed increased expression of nuclear pSTAT5 and Elf5, increased expression of markers of secretory differentiation and milk constituents, and histologically resembled secretory carcinomas of the breast. Overexpression of PTHrP within cells isolated from tumors, but not PTHrP exogenously added to cell culture media, led to activation of STAT5 and milk protein gene expression. In addition, neither ablating the Type 1 PTH/PTHrP receptor (PTH1R) in epithelial cells nor treating Tet-PTHrP;PyMT mice with an anti-PTH1R antibody prevented secretory differentiation or altered tumor latency. These data suggest that PTHrP acts in a cell-autonomous, intracrine manner. Finally, expression of PTHrP in human breast cancers is associated with expression of genes involved in milk production and STAT5 signaling.ConclusionsOur study suggests that PTHrP promotes pathways leading to secretory differentiation and proliferation in both normal mammary epithelial cells and in breast tumor cells.

Cathepsin D deficiency in mammary epithelium transiently stalls breast cancer by interference with mTORC1 signaling

Cathepsin D (CTSD) is a lysosomal protease and a marker of poor prognosis in breast cancer. However, the cells responsible for this association and the function of CTSD in cancer are still incompletely understood. By using a conditional CTSD knockout mouse crossed to the transgenic MMTV-PyMT breast cancer model we demonstrate that CTSD deficiency in the mammary epithelium, but not in myeloid cells, blocked tumor development in a cell-autonomous manner. We show that lack of CTSD impaired mechanistic Target of Rapamycin Complex 1 (mTORC1) signaling and induced reversible cellular quiescence. In line, CTSD-deficient tumors started to grow with a two-month delay and quiescent Ctsd-/- tumor cells re-started proliferation upon long-term culture. This was accompanied by rewiring of oncogenic gene expression and signaling pathways, while mTORC1 signaling remained permanently disabled in CTSD-deficient cells. Together, these studies reveal a tumor cell-autonomous effect of CTSD deficiency, and establish a pivotal role of this protease in the cellular response to oncogenic stimuli.

Metabolome of Mammary Tumors Differs from Normal Mammary Glands But Is Not Altered by Time-restricted Feeding Under Obesogenic Conditions.

Time restricted feeding (TRF) mitigates the high-fat diet-enhanced mammary tumorigenesis in a MMTV-PyMT breast cancer model. We performed untargeted metabolomic and targeted transcriptomic analyses on mammary tumors from MMTV-PyMT mice fed a standard AIN93G diet, a high-fat diet (HFD), or HFD with TRF (12 h, dark phase) and mammary glands from wild-type mice fed the AIN93G diet. The metabolic profile of mammary tumors differed from that of mammary glands; there was no impact of TRF upon tumor metabolome. TRF did reduce elevated expression of Hmgcr, Srebp1, Fads2, and Ppard in mammary tumors, indicating a down-regulation of lipid metabolism. The null effect of TRF on the metabolomic profile does not rule out changes in more refined intracellular signaling pathways. It suggests that the protection of TRF against mammary tumorigenesis may rely upon its action on the host rather than a direct effect on tumor metabolism.

KCa3.1 Channels Confer Radioresistance to Breast Cancer Cells.

KCa3.1 K+ channels reportedly contribute to the proliferation of breast tumor cells and may serve pro-tumor functions in the microenvironment. The putative interaction of KCa3.1 with major anti-cancer treatment strategies, which are based on cytotoxic drugs or radiotherapy, remains largely unexplored. We employed KCa3.1-proficient and -deficient breast cancer cells derived from breast cancer-prone MMTV-PyMT mice, pharmacological KCa3.1 inhibition, and a syngeneic orthotopic mouse model to study the relevance of functional KCa3.1 for therapy response. The KCa3.1 status of MMTV-PyMT cells did not determine tumor cell proliferation after treatment with different concentrations of docetaxel, doxorubicin, 5-fluorouracil, or cyclophosphamide. KCa3.1 activation by ionizing radiation (IR) in breast tumor cells in vitro, however, enhanced radioresistance, probably via an involvement of the channel in IR-stimulated Ca2+ signals and DNA repair pathways. Consistently, KCa3.1 knockout increased survival time of wildtype mice upon syngeneic orthotopic transplantation of MMTV-PyMT tumors followed by fractionated radiotherapy. Combined, our results imply that KCa3.1 confers resistance to radio- but not to chemotherapy in the MMTV-PyMT breast cancer model. Since KCa3.1 is druggable, KCa3.1 targeting concomitant to radiotherapy seems to be a promising strategy to radiosensitize breast tumors.

Abstract 4513: Targeting the DLC1 Rho-GAP tumor suppressor protein to treat solid tumors

Abstract Although cancer usually arises from the combined effects of oncogene activation and tumor suppressor gene inactivation, most targeted cancer treatments are focused on the inhibition of oncoproteins, with less consideration given to the possible concomitant effects on tumor suppressors. Here we describe a critical role of the DLC1 tumor suppressor in the growth regulation of cancer and have identified two kinases that directly phosphorylate DLC1 and attenuate its tumor suppressor activity, leading to the hypothesis that reactivation of DLC1 by the relevant kinase inhibitors should be evaluated as a candidate biomarker of clinical response. We determined that the SRC and AKT kinases directly phosphorylate and attenuate the Rho-GAP and tumor suppressor activities of DLC1 by distinct, cooperative mechanisms and that inhibition of the SRC and/or AKT kinases has strong antitumor activity in two DLC1-positive tumor models that have high SRC and AKT activities, the MMTV-PyMT breast cancer model and tumor xenografts from a NSCLC line. In both models, treatment with the kinase inhibitors results in cooperative DLC1 reactivation, which was monitored by the reactivation of the Rho-GAP and tumor suppressor activities of DLC1. Reactivation of DLC1 makes a critical contribution to the antitumor activity of the AKT or the SRC inhibitors in both models, and the combined treatment with inhibitors of both kinases has even greater Rho-GAP and antitumor activities, which is correlated with greater reductions in RhoA-GTP and its downstream signaling. Furthermore, the combined treatment with AKT and SRC inhibitors induced cellular senescence and apoptosis to a greater degree than single agent treatment, as measured by beta-galactosidase and annexin V expression after short-term tumor treatment. Their antitumor activity is much weaker in isogenic tumors that are DLC1-negative or express DLC1 mutants that are not activatable by the AKT and the SRC inhibitors. A limitation of this therapeutic approach is that it is likely to benefit only those tumors that express moderate to high levels of DLC1 protein. However, several types of solid tumors express DLC1 mRNA but have lost DLC1 protein expression through the epigenetic destabilization of the protein. We hypothesize that it could be beneficial to treat this class of solid tumors with a combination of drugs that can stabilize the DLC1 protein and concurrently inhibit the AKT and SRC activities. Our results indicate the feasibility of this approach, as treatment with proteasome inhibitors in several tumor cell lines that lack detectable DLC1 protein but express its mRNA increased the steady state level of DLC1 protein, which enabled SRC and AKT inhibitors to more potently inhibit tumor cell growth and reduce RhoA-GTP and its signaling. Thus, reactivation of the DLC1 tumor suppressor can be a key therapeutic target, in contrast to the usual situation where the focus is principally on inhibiting pro-oncogenic factors. Citation Format: Brajendra K. Tripathi, Meghan F. Anderman, James H. Doroshow, Douglas R. Lowy. Targeting the DLC1 Rho-GAP tumor suppressor protein to treat solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4513.

Abstract 607: Inhibition of CDK4/6 and Pi3Kγ modulates mammary tumor immune microenvironment to enhance response to immunotherapy

Abstract An antitumor immune response is critical in achieving immunotherapy-driven tumor regression. Despite remarkable clinical advances in immunotherapies, patients with breast cancer have been only moderately responsive. In this study, we tested whether CDK4/6 inhibition in combination with Pi3Kγ inhibition can augment the immunotherapy response. Tumor growth was inhibited in the MMTV-PyMT breast cancer model when treated with palbociclib (CDK4/6i) in combination with anti-CD137, an antibody agonist of T cell co-stimulatory receptor 4-1BB. We also assessed the effect of palbociclib alone in a postsurgical model, where treatment began 5 days after injecting 37,000 MMTV-PyMT cells into the mammary fat pad, and show a significant inhibition of both average tumor weight and volume. In addition, we demonstrate that palbociclib can be effective in reducing total tumor burden in the PyMT-FVB spontaneous breast cancer model. Furthermore, mice treated with palbociclib in this model had an increase in CD3+ cell infiltrate in tumor lung metastasis. We used the Pi3Kγ-null mice treated with palbociclib or vehicle control to assess the effect of dual inhibition of CDK4/6 and Pi3Kγ on MMTV-PyMT tumors. We found an increase in CD45+ cells within the tumor microenvironment and an increase in MHC class II+, antigen-presenting macrophages upon palbociclib treatment. Additionally, gene expression analysis of chemokines in palbociclib-treated MCF-7 cells showed an increased expression of CCL4, CCL5, CXCL9, CXCL10, and CXCL11, each of which is implicated in T-cell tumor-homing. Overall, our results suggest that dual inhibition of CDK4/6 and Pi3Kγ may enhance tumor immunogenicity. Drugs targeting CDK4/6 and Pi3Kγ are of high clinical relevance and our findings indicate that they may be used in combination with immunotherapies for the treatment for breast cancer. Citation Format: Stacey Mont, Kevin Black, Sheau-Chiann Chen, Gregory D. Ayers, Ann Richmond, Anna E. Vilgelm. Inhibition of CDK4/6 and Pi3Kγ modulates mammary tumor immune microenvironment to enhance response to immunotherapy [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 607.

Abstract 3957: SRC inhibitors dephosphorylate and reactivate the DLC1 tumor suppressor: A new biomarker of clinical response

Abstract Although activation of oncogenes and inactivation of tumor suppressor genes frequently play cooperative roles in pathogenesis of cancer, most targeted cancer treatments are focused on oncogene inhibitors, with relatively little consideration given to their concomitant effects on tumor suppressors. In this study, we have identified a direct relationship between the SRC kinase and the tumor suppressor DLC1, determined that SRC phosphorylates and attenuates the tumor suppressor activity of DLC1, and explored whether reactivation of DLC1 may be used as a biomarker for the clinical response to SRC inhibition. Several human cancers have high SRC activity and constitutive up-regulation of RhoA-GTP, which is a pro-oncogenic driver that is implicated in several processes of tumor formation and its progression to metastasis. We determined that SRC directly phosphorylates two Tyrosines in DLC1, Y451 in the linker region and Y701 in the Rho-GAP domain, and inhibits the Rho-GAP activity and tumor suppressor functions of DLC1. SRC phosphorylation of DLC1-Y701 attenuates the hydrolysis of active RhoA-GTP to inactive RhoA-GDP, resulting in an increase in RhoA-GTP levels and its downstream signaling, while SRC phosphorylation of DLC1-Y451 abolishes the binding of tensin to DLC1, which further reduces DLC1 tumor suppressor activity independently of RhoA-GTP. The ability of SRC to phosphorylate its target Tyrosines in DLC1 is facilitated by the ERK-dependent phosphorylation of DLC1-S129, which enhances the binding of the SRC SH3 domain to DLC1 and increases the SRC-dependent phosphorylation of Y451 and Y701 in DLC1. The potentially reversible nature of the SRC phosphorylations of DLC1 may be useful as a biomarker of clinical response. In two models that have high SRC activity and express DLC1 (the mouse MMTV-PyMT breast cancer model and xenografts from a human lung cancer cell line), a 5-day treatment with the SRC inhibitors Saracatanib and Bosutinib have potent antitumor activity (~75% reduction in tumor weight) in conjunction with reducing phosphorylation of SRC target Tyrosines in DLC1 and reactivating DLC1 Rho-GAP activity, which reduces cellular RhoA-GTP levels and its downstream signaling. However, Saracatanib and Bosutinib have much less antitumor activity (~20% reduction in tumor weight) in isogenic versions of the lung cancer xenografts if the SRC inhibitors do not reactivate DLC1, as occurs when DLC1 is not expressed or the SRC phosphorylatable Tyrosines in DLC1 are mutated. Our results highlight the potential importance of DLC1 tumor suppressor reactivation as a biomarker for predicting and monitoring the response to SRC inhibitors. Citation Format: Brajendra K. Tripathi, Meghan Anderman, Xiaolan Qian, Ming Zhou, Dunrui Wang, Alex G. Papageorge, Douglas R. Lowy. SRC inhibitors dephosphorylate and reactivate the DLC1 tumor suppressor: A new biomarker of clinical response [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 3957.

PTEN PDZ-binding domain suppresses mammary carcinogenesis in the MMTV-PyMT breast cancer model

Phosphatase and tension homolog (PTEN) is a potent tumor suppressor that possesses a PDZ-binding domain (PDZ-BD) at the end of its carboxyl terminus, whose functions during tumorigenesis remains unclear. Here, we crossed a mouse strain with germline deletion of PTEN PDZ-BD with MMTV-PyMT breast cancer model, and found that knockout (KO) mice display normal development of mammary glands, but have both increased breast tumorigenicity and lung metastasis. Orthotopic allograft experiments suggest the loss of PTEN PDZ-BD in breast cancer cells rather than in tumor microenvironment plays a prominent role in increasing tumor burden. Through RNA-sequencing, we observed a significant downregulation of myoepithelial marker genes in both KO primary breast cancer and orthotopic allografts. Moreover, these myoepithelial marker genes are significantly downregulated in human breast cancer tissues, and are associated with poorer clinical prognosis. In addition, several homeobox genes were also identified to be downreguated in KO breast cancer, whose expressions showed significant positive correlation with myoepithelial marker genes. Overall, our findings suggest a novel tumor suppressive role of PTEN PDZ-BD in a murine model of breast cancer, and the mechanism involves the dysregulation of homeobox genes which may result in defective myoepithelial differentiation in breast cancer cells.

Myoepithelial cell-specific expression of stefin A as a suppressor of early breast cancer invasion.

Mammography screening has increased the detection of early pre-invasive breast cancers, termed ductal carcinoma in situ (DCIS), increasing the urgency of identifying molecular regulators of invasion as prognostic markers to predict local relapse. Using the MMTV-PyMT breast cancer model and pharmacological protease inhibitors, we reveal that cysteine cathepsins have important roles in early-stage tumorigenesis. To characterize the cell-specific roles of cathepsins in early invasion, we developed a DCIS-like model, incorporating an immortalized myoepithelial cell line (N1ME) that restrained tumor cell invasion in 3D culture. Using this model, we identified an important myoepithelial-specific function of the cysteine cathepsin inhibitor stefin A in suppressing invasion, whereby targeted stefin A loss in N1ME cells blocked myoepithelial-induced suppression of breast cancer cell invasion. Enhanced invasion observed in 3D cultures with N1ME stefin A-low cells was reliant on cathepsin B activation, as addition of the small molecule inhibitor CA-074 rescued the DCIS-like non-invasive phenotype. Importantly, we confirmed that stefin A was indeed abundant in myoepithelial cells in breast tissue. Use of a 138-patient cohort confirmed that myoepithelial stefin A (cystatin A) is abundant in normal breast ducts and low-grade DCIS but reduced in high-grade DCIS, supporting myoepithelial stefin A as a candidate marker of lower risk of invasive relapse. We have therefore identified myoepithelial cell stefin A as a suppressor of early tumor invasion and a candidate marker to distinguish patients who are at low risk of developing invasive breast cancer, and can therefore be spared further treatment. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Abstract 1539: Investigating the mechanisms of tumor suppression mediated by PTEN PDZ-binding domain in a murine breast cancer model

Abstract PTEN (Phosphatase and tensin homolog) functions as a key negative regulator of PI3K-Akt pathway through its lipid phosphatase activity. Mutations or loss of PTEN is frequently observed in brain, breast, prostate and endometrial cancer. PTEN possesses a PDZ-binding domain (PDZ-BD) at the end of its carboxyl terminus. Functionally, PDZ-BD mediates PTEN’s interactions with other PDZ domain-containing proteins, including ZO-1, PSD-95, DLG, etc, which are involved in the formation of cell junctions, post-synaptic densities, and serves as scaffolding proteins for multiple signaling complexes. However, whether the PTEN PDZ-binding domain is required to suppress tumorigenesis still remains obscure. To study the role of PTEN PDZ-BD in breast epithelial tumorigenesis, a PTEN-ΔPDZ mice lacking this domain was generated. PTEN expression level and activity of PI3K-Akt pathway are not changed in the mammary epithelial cells, indicating that PDZ-BD is dispensable for PTEN stability and negative regulation of the PI3K-Akt pathway. Further, we crossed the PTEN-ΔPDZ mice with the MMTV-PyMT breast cancer model and observed increased total tumor burden as well as higher tendency of lung metastasis in PTEN-ΔPDZ mice. This data indicates that PTEN PDZ-BD is indeed important for tumor suppression during breast cancer progression. Previously, PTEN has been reported to govern the integrity of tight junctions and apical-basal polarity of epithelial cells, which requires PTEN’s interactions with other proteins through the PDZ-BD. However, through in vitro three-dimensional culture of primary mouse mammary epithelial cells, we failed to observe any disruption of either apical-basal polarity or tight junction. Meanwhile, we investigated the alteration of the transcriptome of breast cancer tissue in PTEN-ΔPDZ mice by performing RNA-sequencing. Several enriched signaling pathways were identified by Ingenuity Pathway Analysis (IPA), which seems to be mainly associated with cell skeleton signaling as well as signaling interaction between extracellular matrix and the cancer cell. A list of genes were validated by RT-PCR, and functional validation of these genes during the tumorigenesis in our animal model is to be carried out in the near future. This work was supported by a General Research Fund grant (#460713) from the Hong Kong University Grants Committee to AC. YM was supported by a Hong Kong PhD Fellowship (PF12-13584), and a Lo Kwee-Seong Biomedical Research Seed Fund to AC. Citation Format: Mingfei Yan, Alexander Many, Hong Guan, Penelope M. Or, Andrew M. Chan. Investigating the mechanisms of tumor suppression mediated by PTEN PDZ-binding domain in a murine breast cancer model [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 1539. doi:10.1158/1538-7445.AM2017-1539

Abstract 253: Mammary tumorigenesis causes bone loss and dietary selenium supplementation does not affect such bone loss in male MMTV-PyMT mice

Abstract Cancer progression is accompanied by wasting that eventually results in cachexia characterized by significant weight loss and multi-organ functional failures. Limited clinical trials indicate that bone is adversely affected by cancer-associated wasting. To determine the effects of breast cancer on skeletal health, we performed micro-computed tomographic analysis of femurs and vertebrae collected from a recently completed study showing that dietary supplementation with selenium (methylseleninic acid, 2.5 mg Se/kg) reduced male mammary tumorigenesis in MMTV-PyMT mice. Compared to age-matched non-tumor-bearing mice (MMTV-PyMT negative), the presence of mammary tumors significantly reduced bone volume fraction, trabecular thickness and bone mineral density and increased the structure model index (an indicator of the plate- and rod-like geometry of trabecular structure) in femoral trabecular bone. Mammary tumor development did not affect vertebral trabeculae nor femoral and vertebral cortical bone, except it significantly reduced cortical bone thickness of vertebrae. There were no differences in aforementioned measurements between groups with or without selenium supplementation. In conclusion, mammary tumorigenesis causes bone loss and dietary selenium supplementation at 2.5 mg Se/kg, which is antitumorigenic, does not affect mammary tumor-associated bone loss in this male MMTV-PyMT breast cancer model. Citation Format: Lin Yan. Mammary tumorigenesis causes bone loss and dietary selenium supplementation does not affect such bone loss in male MMTV-PyMT mice [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 253. doi:10.1158/1538-7445.AM2017-253

Mint3 in bone marrow-derived cells promotes lung metastasis in breast cancer model mice

Breast cancer is one of the most common cancers in women in the world. Although breast cancer is well treatable at the early stage, patients with distant metastases show a poor prognosis. Data from recent studies using transplantation models indicate that Mint3/APBA3 might promote breast cancer malignancy. However, whether Mint3 indeed contributes to tumor development, progression, or metastasis in vivo remains unclear. To address this, here we examined whether Mint3 depletion affects tumor malignancy in MMTV-PyMT breast cancer model mice. In MMTV-PyMT mice, Mint3 depletion did not affect tumor onset and tumor growth, but attenuated lung metastases. Experimental lung metastasis of breast cancer Met-1 cells derived from MMTV-PyMT mice also decreased in Mint3-depleted mice, indicating that host Mint3 expression affected lung metastasis of MMTV-PyMT-derived breast cancer cells. Further bone marrow transplant experiments revealed that Mint3 in bone marrow-derived cells promoted lung metastasis in MMTV-PyMT mice. Thus, targeting Mint3 in bone marrow-derived cells might be a good strategy for preventing metastasis and improving the prognosis of breast cancer patients.

Abstract 1627: Kinase-independent function of focal adhesion kinase in lung metastasis of breast cancer

Abstract Focal adhesion kinase (FAK) has been shown to promote mammary tumorigenesis, lung metastasis and the maintenance of cancer stem cells in the MMTV-PyMT breast cancer model. Using mammary epithelial cell (MaEC)-specific FAK knockout and kinase-defective (KD) knockin mouse models, we recently demonstrated that loss of FAK kinase activity in MaECs specifically impaired luminal progenitor proliferation and alveologenesis, whereas FAK kinase-independent function was sufficient to support ductal invasion and basal mammary stem cell activity. To further elucidate this kinase-independent role of FAK in mammary tumorigenesis and lung metastasis, we established and analyzed three cohorts of PyMT mice expressing wild type FAK (Ctl-MT), KD-FAK (cKD-MT) and FAK-null (cKO-MT) in MaECs. This study revealed that both FAK deletion and KD knockin mutation in MaECs significantly suppressed mammary tumorigenesis and the content of stem-like tumor cells (CD24+CD29+CD61+). However, in contrast to cKO-MT mice that displayed diminished metastatic nodule formation in the lungs, loss of FAK kinase activity by KD knockin mutation did not affect lung metastatic nodule formation. Using a Boyden chamber assay, we further demonstrated that, unlike cKO-MT tumor cells that displayed diminished cell migration induced by fibronectin or TGFβ1 (as compared to Ctl-MT tumor cells), cKD-MT tumor cells exhibited strong fibronectin-, but not TGFβ1-induced migratory activity. Immunofluorescence of primary tumor sections also revealed that both Ctl-MT and cKD-MT tumors displayed strong ERK activation at tumor periphery, whereas cKO-MT tumors had weak ERK activation in this region. Lastly, when treated with or without paclitaxel in culture, we found that paclitaxel was able to induce strong expression of cleaved caspase 3 and p21 in cKO-MT but not Ctl-MT and cKD-MT tumor cells, suggesting a role for FAK kinase-independent function in the suppression of p21 expression and protection of apoptosis. Together, this study strongly implicates a role for FAK kinase-independent function in promoting lung metastasis. Citation Format: Ming Luo, Max S. Wicha, Jun-Lin Guan. Kinase-independent function of focal adhesion kinase in lung metastasis of breast cancer. [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 1627.

Abstract 4377: AKT positively regulates Rho-GTP by attenuating the GAP activity of the DLC1 tumor suppressor: a mechanistic study with translational implications

Abstract Many human cancers have high AKT activity and constitutive up-regulation of Rho-GTP, which is a major factor in the neoplastic process, but AKT is not known to regulate Rho-GTP. In this study, we show that AKT positively regulates Rho-GTP by directly phosphorylating tumor suppressor DLC1 and attenuating the Rho-GAP activity of DLC1, which catalyzes the conversion of active Rho-GTP to inactive Rho-GDP. Interestingly, AKT was found to increase Rho-GTP and its downstream activities in DLC1-positive cancer lines whether the DLC1 was endogenous or transfected, but not in DLC1-negative lines. Similarly, when epithelial cell lines were stimulated with EGF, they activated AKT, which increased Rho-GTP in a DLC1-dependent manner. Three Serines (S298, S329, S567) in DLC1 have AKT consensus motifs and were phosphorylated by AKT in vitro and in vivo. Their phosphorylation attenuated the Rho-GAP and tumor suppressor activities of DLC1 - including decreased cell migration, focal adhesion turnover, anchorage-independent growth, and tumor growth in mice - as did the combined phosphomimetic mutant DLC1-3D. By contrast, the combined Serine to Alanine mutant DLC1-3A was even more active than wild type DLC1 (DLC1-WT). Remarkably, an AKT inhibitor stimulated the tumor suppressor and Rho-GAP activities of DLC1-WT, but did not influence DLC1-3A or DLC1-3D. The N-terminal half of DLC1 (amino acids 1-600) bound the DLC1 Rho-GAP domain (amino acids 609-878), as determined by in vivo complex formation and by microscale thermophoresis, which measures protein interactions in close-to-native conditions. There was increased binding and decreased Rho-GAP activity when the three Serines in the N-terminus were phosphorylated or carried the 3D mutations. Conversely, there was decreased binding and increased Rho-GAP activity when the Serines were not phosphorylated or carried the 3A mutations. In a xenograft mouse model, AKT inhibitor treatment of DLC1-positive palpable tumors reduced their size, their Rho-GTP level, and their Rho-dependent signaling, but did not affect these three parameters in isogenic DLC1-negative tumors. Similarly, in the MMTV-PyMT breast cancer model, which had high AKT activity, high Rho-GTP, and expressed DLC1, an AKT inhibitor reduced the size of palpable tumors, and reduced Rho-GTP and its downstream signaling. AKT inhibition reduced DLC1 phosphorylation in both tumor models. We conclude: AKT can increase Rho-GTP by phosphorylating three N-terminal Serines in DLC1, which attenuates its Rho-GAP and tumor suppressor functions; the N-terminus of DLC1 is an auto-inhibitory domain that reversibly binds the Rho-GAP domain; AKT attenuates DLC1 functions by phosphorylating the Serines in the N-terminus, which increases its binding the Rho-GAP domain; and AKT inhibition has greater anti-tumor activity in DLC1-positive tumors than in DLC1-negative tumors. Citation Format: Brajendra K. Tripathi, Tiera Grant, Philipp Mertins, Xiaolan Qian, Dunrui Wang, Alex G. Papageorge, Steven A. Carr, Douglas R. Lowy. AKT positively regulates Rho-GTP by attenuating the GAP activity of the DLC1 tumor suppressor: a mechanistic study with translational implications. [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 4377.

Tumour stroma-derived lipocalin-2 promotes breast cancer metastasis.

Tumour cell-secreted factors skew infiltrating immune cells towards a tumour-supporting phenotype, expressing pro-tumourigenic mediators. However, the influence of lipocalin-2 (Lcn2) on the metastatic cascade in the tumour micro-environment is still not clearly defined. Here, we explored the role of stroma-derived, especially macrophage-released, Lcn2 in breast cancer progression. Knockdown studies and neutralizing antibody approaches showed that Lcn2 contributes to the early events of metastasis in vitro. The release of Lcn2 from macrophages induced an epithelial-mesenchymal transition programme in MCF-7 breast cancer cells and enhanced local migration as well as invasion into the extracellular matrix, using a three-dimensioanl (3D) spheroid model. Moreover, a global Lcn2 deficiency attenuated breast cancer metastasis in both the MMTV-PyMT breast cancer model and a xenograft model inoculating MCF-7 cells pretreated with supernatants from wild-type and Lcn2-knockdown macrophages. To dissect the role of stroma-derived Lcn2, we employed an orthotopic mammary tumour mouse model. Implantation of wild-type PyMT tumour cells into Lcn2-deficient mice left primary mammary tumour formation unaltered, but specifically reduced tumour cell dissemination into the lung. We conclude that stroma-secreted Lcn2 promotes metastasis in vitro and in vivo, thereby contributing to tumour progression. Our study highlights the tumourigenic potential of stroma-released Lcn2 and suggests Lcn2 as a putative therapeutic target. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Abstract 308: The role of PTEN PDZ-binding domain in mammary gland tumorigenesis

Abstract PTEN functions as a key negative regulator of PI3K-Akt pathway through its lipid phosphatase activity. Mutations or loss of PTEN is frequently observed in brain, breast, prostate and endometrial cancer. PTEN also possesses a PDZ-binding domain (PDZ-BD) at its carboxyl terminus, which can be recruited by PDZ-containing proteins like ZO-1 to cell tight junction. Therefore, PTEN may have a potential role in regulating epithelial tight junction integrity. To study the role of PTEN PDZ-BD in breast epithelial tumorigenesis, a PTEN-ΔPDZ mice lacking this domain was generated and further crossed with MMTV-PyMT breast cancer model. As a result, PTENΔPDZ/MMTV-PyMT mice show enhanced tumor growth and metastasis. To elucidate tumor suppressive mechanisms of PTEN PDZ-BD, three-dimensional cultures of PTENΔPDZ mouse mammary epithelial cells (MMEs) was carried out. Though PTEN-ΔPDZ MMEs spheres have normal morphogenesis in 3D culture, they possess larger diameters than wild-type (WT). Besides, both tight junction (ZO-1) and adherent junction (E-Cadherin) of MMEs were examined in 3D culture, though no major differences in cell junction and polarity was observed between PTEN-ΔPDZ and PTEN-WT. Future work will study signaling activities of PTEN-ΔPDZ/MMTV-PyMT breast cancer cells, morphogenesis, polarity and cell junction of PTEN-ΔPDZ/MMTV-PyMT MMEs in 3D cultures. This work was supported by NIH RO1 (CA133669), Hong Kong Research Grant Council (460713) and Hong Kong PhD Fellowship Scheme (PF12-13584) Citation Format: Mingfei Yan, Penelope Or, Andrew Chan. The role of PTEN PDZ-binding domain in mammary gland tumorigenesis. [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 308. doi:10.1158/1538-7445.AM2015-308

Spontaneous lung and lymph node metastasis in transgenic breast cancer is independent of the urokinase receptor uPAR.

Urokinase-type plasminogen activator (uPA) is an extracellular protease that plays a pivotal role in tumor progression. uPA activity is spatially restricted by its anchorage to high-affinity uPA receptors (uPAR) at the cell surface. High tumor tissue expression of uPA and uPAR is associated with poor prognosis in lung, breast, and colon cancer patients in clinical studies. Genetic deficiency of uPA leads to a significant reduction in metastases in the murine transgenic MMTV-PyMT breast cancer model, demonstrating a causal role for uPA in cancer dissemination. To investigate the role of uPAR in cancer progression, we analyze the effect of uPAR deficiency in the same cancer model. uPAR is predominantly expressed in stromal cells in the mouse primary tumors, similar to human breast cancer. In a cohort of MMTV-PyMT mice [uPAR-deficient (n = 31) or wild type controls (n = 33)], tumorigenesis, tumor growth, and tumor histopathology were not significantly affected by uPAR deficiency. Lung and lymph node metastases were also not significantly affected by uPAR deficiency, in contrast to the significant reduction seen in uPA-deficient mice. Taken together, our data show that the genetic absence of uPAR does not influence the outcome of the MMTV-PyMT cancer model.

Novel nano-sized MR contrast agent mediates strong tumor contrast enhancement in an oncogene-driven breast cancer model.

The current study was carried out to test the potential of a new nanomaterial (Spago Pix) as a macromolecular magnetic MR contrast agent for tumor detection and to verify the presence of nanomaterial in tumor tissue. Spago Pix, synthesized by Spago Nanomedical AB, is a nanomaterial with a globular shape, an average hydrodynamic diameter of 5 nm, and a relaxivity (r1) of approximately 30 (mM Mn)−1 s−1 (60 MHz). The material consists of an organophosphosilane hydrogel with strongly chelated manganese (II) ions and a covalently attached PEG surface layer. In vivo MRI of the MMTV-PyMT breast cancer model was performed on a 3 T clinical scanner. Tissues were thereafter analyzed for manganese and silicon content using inductively coupled plasma-atomic emission spectroscopy (ICP-AES). The presence of nanomaterial in tumor and muscle tissue was assessed using an anti-PEG monoclonal antibody. MR imaging of tumor-bearing mice (n = 7) showed a contrast enhancement factor of 1.8 (tumor versus muscle) at 30 minutes post-administration. Contrast was retained and further increased 2–4 hours after administration. ICP-AES and immunohistochemistry confirmed selective accumulation of nanomaterial in tumor tissue. A blood pharmacokinetics analysis showed that the concentration of Spago Pix gradually decreased over the first hour, which was in good agreement with the time frame in which the accumulation in tumor occurred. In summary, we demonstrate that Spago Pix selectively enhances MR tumor contrast in a clinically relevant animal model. Based on the generally higher vascular leakiness in malignant compared to benign tissue lesions, Spago Pix has the potential to significantly improve cancer diagnosis and characterization by MRI.

Abstract P6-01-01: Diet-induced obesity increases tumor growth and promotes angiogenesis in a murine model of breast cancer

Abstract Recent epidemiological studies provide strong evidence suggesting obesity is a risk factor in several cancers, including breast cancer. However, the exact molecular mechanisms by which obesity increases the risk of breast cancer are poorly understood. In this study, we evaluated the effect of diet-induced obesity on breast carcinogenesis in the MMTV-PyMT breast cancer model. Four-week old, female mice (n = 40) were randomized to high fat (HFD) or low fat (LFD) diets for 8 weeks. Body weights were obtained weekly and mice were imaged for mammary tumor volumes by 3D ultrasound. Tumor burden was collected during early and late stages, weighed and fixed for immunohistochemical staining. Blood and tissue from the liver, lung, and mesenteric fat were also collected. We also compared the basal VEGF protein expression in skeletal muscle, adipose tissue and mammary tumors from MMTV-PyMT mice and compared these levels against background nontransgenic FVB/N mice. Average tumor volumes and tumor burdens were markedly greater in the mice fed HFD than in LFD and showed an increased number of metastases to the lung by 2-fold. HFD increased mammary cell proliferation and elevated Chemokine (C-C motif) ligand 2 (CCL2) in the blood, tumor beds and adipose tissue. Histopathological analysis showed more tumor-associated macrophages and increased microvessel density with diet-induced obesity. Significantly more crown-like structures were evident in adipose tissue from obese mice at both early and late stage carcinoma. Adipose tissue of MMTV-PyMT mice exhibited greater VEGF protein expression compared to adipose tissue of control FVB mice, and greater VEGF protein compared to tumor VEGF expression. Our findings demonstrate that diet-induced obesity exacerbates breast cancer progression in MMTV-PyMT mice and suggests that high body adiposity caused by diet-induced changes promotes an inflammatory and angiogenic phenotype in breast cancer. NIH P20 RR016440, P30 R032138 and GM103488, and P20 RR016477. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-01-01.

Tumor Microenvironment Regulates Metastasis and Metastasis Genes of Mouse MMTV-PymT Mammary Cancer Cells In Vivo

Metastasis is the primary cause of death in breast cancer patients, yet there are challenges to modeling this process in vivo. The goal of this study was to analyze the effects of injection site on tumor growth and metastasis and gene expression of breast cancer cells in vivo using the MMTV-PymT breast cancer model (Met-1 cells). Met-1 cells were injected into 5 sites (subcutaneous, mammary fat pad, tail vein, intracardiac, and intratibial), and tumors and metastases were monitored using bioluminescent imaging and confirmed with gross necropsy and histopathology. Met-1 tumors were analyzed based on morphology and changes in gene expression in each tissue microenvironment. There were 6 permissible sites of Met-1 tumor growth (mammary gland, subcutis, lung, adrenal gland, ovary, bone). Met-1 cells grew faster in the subcutis compared to mammary fat pad tumors (highest Ki-67 index). Morphologic differences were evident in each tumor microenvironment. Finally, 7 genes were differentially expressed in the Met-1 tumors in the 6 sites of growth or metastasis. This investigation demonstrates that breast cancer progression and metastasis are regulated by not only the tumor cells but also the experimental model and unique molecular signals from the tumor microenvironment.