Elevated MET receptor tyrosine kinase correlates with poor outcome in breast cancer, yet the reasons for this are poorly understood. We thus generated a transgenic mouse model targeting expression of an oncogenic Met receptor (Met mt ) to the mammary epithelium. We show that Met mt induces mammary tumors with multiple phenotypes. These reflect tumor subtypes with gene expression and immunostaining profiles sharing similarities to human basal and luminal breast cancers. Within the basal subtype, Met mt induces tumors with signatures of WNT and epithelial to mesenchymal transition (EMT). Among human breast cancers, MET is primarily elevated in basal and ERBB2-positive subtypes with poor prognosis, and we show that MET, together with EMT marker, SNAIL, are highly predictive of poor prognosis in lymph nodenegative patients. By generating a unique mouse model in which the Met receptor tyrosine kinase is expressed in the mammary epithelium, along with the examination of MET expression in human breast cancer, we have established a specific link between MET and basal breast cancer. This work identifies basal breast cancers and, additionally, poor-outcome breast cancers, as those that may benefit from anti-MET receptor therapies.gene expression profiling ͉ mouse models ͉ epithelial to mesenchymal transition B reast cancer is a heterogeneous disease that comprises distinct biological entities that are correlated with diverse clinical outcomes and responses to treatment. Gene expression profiling and molecular pathology have revealed that breast cancers naturally divide into the luminal, ERBB2-positive, and basal-like subtypes (1, 2). These subtypes were named to reflect gene expression patterns of the 2 principal cell types of the differentiated breast, luminal epithelial cells lining the duct and lobule, and myoepithelial cells that form a single layer surrounding the luminal cells. The luminal subtype comprises ϳ60% of breast cancers, is estrogen receptor (ESR1)-positive, and expresses ESR1-responsive genes and luminal markers such as keratin 8/18. Up to 25% of breast cancers are identified with overexpression/amplification of the ERBB2 receptor tyrosine kinase, and these tumors are generally ESR1/ progesterone receptor (PGR)-negative. The basal group is characterized as ESR1/PGR/ERBB2-negative and is frequently positive for basal keratins 5/6 (3, 4). Breast cancers within the luminal subtype receive antiestrogen therapies and tend to have a good prognosis. Because of the lack of treatment options, patients within the basal subtype historically have a poor prognosis (1). Hence, an understanding of the signaling pathways active in these tumors is crucial for the generation of targeted therapies.The MET receptor tyrosine kinase, which is the receptor for hepatocyte growth factor/scatter factor (HGF/SF), is expressed at elevated levels in 15-20% of human breast cancers (5), and is a prognostic factor for poor outcome (6, 7). High levels of the MET receptor ligand HGF/SF in the serum of breast cancer patients is also correl...

Prolactin hormone (PRL) is well characterized as a terminal differentiation factor for mammary epithelial cells and as an autocrine growth/survival factor in breast cancer cells. However, this function of PRL may not fully signify its role in breast tumorigenesis. Cancer is a complex multistep progressive disease resulting not only from defects in cell growth but also in cell differentiation. Indeed, dedifferentiation of tumor cells is now recognized as a crucial event in invasion and metastasis. PRL plays a critical role in inducing/maintaining differentiation of mammary epithelial cells, suggesting that PRL signaling could serve to inhibit tumor progression. We show here that in breast cancer cells, PRL and Janus-activated kinase 2, a major kinase involved in PRL signaling, play a critical role in regulating epithelial-mesenchymal transformation (EMT), an essential process associated with tumor metastasis. Activation of the PRL receptor (PRLR), achieved by restoring PRL/JAK2 signaling in mesenchymal-like breast cancer cells, MDA-MB-231, suppressed their mesenchymal properties and reduced their invasive behavior. While blocking PRL autocrine function in epithelial-like breast cancer cells, T47D, using pharmacologic and genetic approaches induced mesenchymal-like phenotypic changes and enhanced their invasive propensity. Moreover, our results indicate that blocking PRL signaling led to activation of mitogen-activated protein kinase (extracellular signal-regulated kinase 1/2) and transforming growth factor-B/Smad signaling pathways, two major prometastatic pathways. Furthermore, our results indicate that following PRL/JAK2 inhibition, ERK1/2 activation precedes and is required for Smad2 activation and EMT induction in breast cancer cells. Together, these results highlight PRL as a critical regulator of epithelial plasticity and implicate PRL as an invasion suppressor hormone in breast cancer. (Cancer Res 2006; 66(3): 1824-32)

Triple-negative breast cancer (TNBC) accounts for ∼20% of cases and contributes to basal and claudin-low molecular subclasses of the disease. TNBCs have poor prognosis, display frequent mutations in tumor suppressor gene p53 (TP53), and lack targeted therapies. The MET receptor tyrosine kinase is elevated in TNBC and transgenic Met models (Met mt ) develop basal-like tumors. To investigate collaborating events in the genesis of TNBC, we generated Met mt mice with conditional loss of murine p53 (Trp53) in mammary epithelia. Somatic Trp53 loss, in combination with Met mt , significantly increased tumor penetrance over Met mt or Trp53 loss alone. Unlike Met mt tumors, which are histologically diverse and enriched in a basal-like molecular signature, the majority of Met mt tumors with Trp53 loss displayed a spindloid pathology with a distinct molecular signature that resembles the human claudin-low subtype of TNBC, including diminished claudins, an epithelial-tomesenchymal transition signature, and decreased expression of the microRNA-200 family. Moreover, although mammary specific loss of Trp53 promotes tumors with diverse pathologies, those with spindloid pathology and claudin-low signature display genomic Met amplification. In both models, MET activity is required for maintenance of the claudin-low morphological phenotype, in which MET inhibitors restore cell-cell junctions, rescue claudin 1 expression, and abrogate growth and dissemination of cells in vivo. Among human breast cancers, elevated levels of MET and stabilized TP53, indicative of mutation, correlate with highly proliferative TNBCs of poor outcome. This work shows synergy between MET and TP53 loss for claudin-low breast cancer, identifies a restricted claudin-low gene signature, and provides a rationale for anti-MET therapies in TNBC.Met RTK | EMT | mouse model | gene expression

The protein tyrosine phosphatase SHP-2 is an important regulator of the Janus kinase-2 (Jak2)/signal transducer and activator of transcription (Stat) pathway downstream of the cytokine/prolactin receptor family. We report that SHP-2 dephosphorylates tyrosine (Tyr-1007) of Jak2 kinase, a critical recruitment site for the ubiquitin ligase-associated inhibitory protein suppressor of cytokine signaling-1 (SOCS-1), thereby contributing to Jak2 stability. Inactivation of SHP-2 function by blocking receptor/SHP-2 association or by using a catalytically inactive mutant of SHP-2 led to a marked increase in Jak2 ubiquitination/degradation, Jak2 phosphorylation on Tyr-1007, and Jak2/SOCS-1 association. Furthermore, functional studies indicate that modulating the interaction of Jak2/SOCS-1 by SHP-2 is essential for prolactin/Stat5-mediated signaling. Together our results provide a novel function for SHP-2 as a positive regulator of cytokine receptor signaling by regulating ubiquitination/degradation pathways.

The Src homology 2 (SH2) domain containing proteintyrosine phosphatase SHP-2 contributes to prolactin receptor (PRLR) signal transduction to ␤-casein gene promoter activation. We report for the first time that SHP-2 physically associates with the signal transducer and activator of transcription-5a (Stat5a), an important mediator of PRLR signaling to milk protein gene activation, in the mouse mammary HC11 and the human breast cancer T47D cells when stimulated with prolactin (PRL) and human growth hormone, respectively. In addition, overexpression studies indicate that the carboxyl-terminal SH2 domain of SHP-2 is required to maintain tyrosine phosphorylation of Stat5 and its interaction with SHP-2. Furthermore, we demonstrate by nuclear co-immunoprecipitation and indirect immunofluorescence studies that PRL stimulation of mammary cells leads to the nuclear translocation of SHP-2 as a complex with Stat5a. This process was found to involve the catalytic activity of the phosphatase. Finally, using the Stat5 GAS (␥-activated sequence) element of the ␤-casein gene promoter in electrophoretic mobility shift assays, we demonstrate that PRL induces the SHP-2-Stat5a complex to bind to DNA. The presence of the phosphatase in the protein-bound DNA complex was verified by using polyclonal antisera to SHP-2. Our studies indicate a tight physical and functional interaction between SHP2 and Stat5 required for regulation and perpetuation of PRLmediated signaling in mammary cells and suggest a potential role for SHP-2 in the nucleus.

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Invadopodia are membrane protrusions used by cancer cells to remodel and invade the extracellular matrix. Here, Rajadurai et al. show that the lipid phosphatase SHIP2 recruits the Ena/VASP-family actin regulatory protein Mena to stabilize invadopodia membrane protrusions and promote cell invasion.

When expressed alone at high levels, the human adenovirus E4orf4 protein exhibits tumor cell-specific p53-independent toxicity. A major E4orf4 target is the B55 class of PP2A regulatory subunits, and we have shown recently that binding of E4orf4 inhibits PP2A B55 phosphatase activity in a dose-dependent fashion by preventing access of substrates (M. Z. Mui et al., PLoS Pathog 9:e1003742, 2013, http://dx.doi.org/10.1371/journal.ppat.1003742). While interaction with B55 subunits is essential for toxicity, E4orf4 mutants exist that, despite binding B55 at high levels, are defective in cell killing, suggesting that other essential targets exist. In an attempt to identify additional targets, we undertook a proteomics approach to characterize E4orf4-interacting proteins. Our findings indicated that, in addition to PP2A B55 subunits, ASPP-PP1 complex subunits were found among the major E4orf4-binding species. Both the PP2A and ASPP-PP1 phosphatases are known to positively regulate effectors of the Hippo signaling pathway, which controls the expression of cell growth/survival genes by dephosphorylating the YAP transcriptional coactivator. We find here that expression of E4orf4 results in hyperphosphorylation of YAP, suggesting that Hippo signaling is affected by E4orf4 interactions with PP2A B55 and/or ASPP-PP1 phosphatases. Furthermore, knockdown of YAP1 expression was seen to enhance E4orf4 killing, again consistent with a link between E4orf4 toxicity and inhibition of the Hippo pathway. This effect may in fact contribute to the cancer cell specificity of E4orf4 toxicity, as many human cancer cells rely heavily on the Hippo pathway for their enhanced proliferation. IMPORTANCEThe human adenovirus E4orf4 protein has been known for some time to induce tumor cell-specific death when expressed at high levels; thus, knowledge of its mode of action could be of importance for development of new cancer therapies. Although the B55 form of the phosphatase PP2A has long been known as an essential E4orf4 target, genetic analyses indicated that others must exist. To identify additional E4orf4 targets, we performed, for the first time, a large-scale affinity purification/mass spectrometry analysis of E4orf4 binding partners. Several additional candidates were detected, including key regulators of the Hippo signaling pathway, which enhances cell viability in many cancers, and results of preliminary studies suggested a link between inhibition of Hippo signaling and E4orf4 toxicity.