Syngeneic mouse model of human HER2+ metastatic breast cancer for the evaluation of trastuzumab emtansine combined with oncolytic rhabdovirus

BackgroundEstablished mouse models of HER2+ cancer are based on the over-expression of rodent Neu/Erbb2 homologues, which are incompatible with human HER2 (huHER2) targeted therapeutics. Additionally, the use of immune-deficient xenograft or transgenic models precludes assessment of native anti-tumour immune responses. These hurdles have been a challenge for our understanding of the immune mechanisms behind huHER2-targeting immunotherapies.MethodsTo assess the immune impacts of our huHER2-targeted combination strategy, we generated a syngeneic mouse model of huHER2+ breast cancer, using a truncated form of huHER2, HER2T. Following validation of this model, we next treated tumour-bearing with our immunotherapy strategy: oncolytic vesicular stomatitis virus (VSVΔ51) with clinically approved antibody-drug conjugate targeting huHER2, trastuzumab emtansine (T-DM1). We assessed efficacy through tumour control, survival, and immune analyses.ResultsThe generated truncated HER2T construct was non-immunogenic in wildtype BALB/c mice upon expression in murine mammary carcinoma 4T1.2 cells. Treatment of 4T1.2-HER2T tumours with VSVΔ51+T-DM1 yielded robust curative efficacy compared to controls, and broad immunologic memory. Interrogation of anti-tumour immunity revealed tumour infiltration by CD4+ T cells, and activation of B, NK, and dendritic cell responses, as well as tumour-reactive serum IgG.ConclusionsThe 4T1.2-HER2T model was used to evaluate the anti-tumour immune responses following our complex pharmacoviral treatment strategy. These data demonstrate utility of the syngeneic HER2T model for assessment of huHER2-targeted therapies in an immune-competent in vivo setting. We further demonstrated that HER2T can be implemented in multiple other syngeneic tumour models, including but not limited to colorectal and ovarian models. These data also suggest that the HER2T platform may be used to assess a range of surface-HER2T targeting approaches, such as CAR-T, T-cell engagers, antibodies, or even retargeted oncolytic viruses

Activated Rac1 requires gp130 for Stat3 activation, cell proliferation and migration

Rac1 (Rac) is a member of the Rho family of small GTPases which controls cell migration by regulating the organization of actin filaments. Previous results suggested that mutationally activated forms of the Rho GTPases can activate the Signal Transducer and Activator of Transcription-3 (Stat3), but the exact mechanism is a matter of controversy. We recently demonstrated that Stat3 activity of cultured cells increases dramatically following E-cadherin engagement. To better understand this pathway, we now compared Stat3 activity levels in mouse HC11 cells before and after expression of the mutationally activated Rac1 (RacV12), at different cell densities. The results revealed for the first time a dramatic increase in protein levels and activity of both the endogenous Rac and RacV12 with cell density, which was due to inhibition of proteasomal degradation. In addition, RacV12-expressing cells had higher Stat3, tyrosine-705 phosphorylation and activity levels at all densities, indicating that RacV12 is able to activate Stat3. Further examination of the mechanism of Stat3 activation showed that RacV12 expression caused a surge in mRNA of Interleukin-6 (IL6) family cytokines, known potent Stat3 activators. Knockdown of gp130, the common subunit of this family reduced Stat3 activity, indicating that these cytokines may be responsible for the Stat3 activation by RacV12. The upregulation of IL6 family cytokines was required for cell migration and proliferation induced by RacV12, as shown by gp130 knockdown experiments, thus demonstrating that the gp130/Stat3 axis represents an essential effector of activated Rac for the regulation of key cellular functions

Differential effects of c-Ras upon transformation, adipocytic differentiation, and apoptosis mediated by the simian virus 40 large tumor antigen

To investigate the functional relationship between the ability of the simian virus 40 large tumor antigen (TAg) to transform and its ability to block adipocytic differentiation and induce apoptosis, we expressed TAg in C3H10T1/2 (10T1/2)-derived preadipocytes. The results demonstrated that differentiation could be suppressed at lower TAg levels than at the levels required for full neoplastic conversion. Progressively higher TAg levels were accompanied by apoptosis induction in this system. To further examine the role of the cellular Ras protooncogene product (Ras) in TAg function, TAg was expressed in 10T1/2-derived preadipocytes rendered deficient in Ras activity by transfection with inducible or constitutive antisense ras gene constructs. The results indicated that Ras is required for TAg-mediated transformation and for suppression of adipocytic differentiation, while TAg-mediated apoptosis following serum starvation was independent from Ras action. Unexpectedly, our results further demonstrated a dramatic reduction in the levels of the TAg protein itself as differentiation progressed in Ras-knockdown cells, with a concomitant reduction in TAg’s ability to induce apoptosis as a result. These findings suggest that Ras, although cytoplasmic, is an integral component of the pathway whereby TAg, an oncoprotein believed to have primarily nuclear targets, suppresses differentiation or induces neoplastic conversion of murine preadipocytes

Stat3 and Gap Junctions in Normal and Lung Cancer Cells

Gap junctions are channels linking the interiors of neighboring cells. A reduction in gap junctional intercellular communication (GJIC) correlates with high cell proliferation, while oncogene products such as <i>Src</i> suppress GJIC, through the Ras/Raf/Erk and other effector pathways. High <i>Src</i> activity was found to correlate with high levels of the <i>Src</i> effector, Signal Transducer and Activator of Transcription-3 (Stat3) in its tyrosine-705 phosphorylated, <i>i.e.</i>, transcriptionally activated form, in the majority of Non-Small Cell Lung Cancer lines examined. However, Stat3 inhibition did not restore GJIC in lines with high <i>Src</i> activity. In the contrary, Stat3 inhibition in normal cells or in lines with low <i>Src</i> activity and high GJIC eliminated gap junctional communication. Therefore, despite the fact that Stat3 is growth promoting and in an activated form acts like an oncogene, it is actually required for junctional permeability

The R(H)Oads To Stat3: Stat3 Activation By The Rho Gtpases

The signal transducer and activator of transcription-3 (Stat3) is a member of the STAT family of cytoplasmic transcription factors. Overactivation of Stat3 is detected with high frequency in human cancer and is considered a molecular abnormality that supports the tumor phenotype. Despite concerted investigative efforts, the molecular mechanisms leading to the aberrant Stat3 activation and Stat3-mediated transformation and tumorigenesis are still not clearly defined. Recent evidence reveals a crosstalk close relationship between Stat3 signaling and members of the Rho family of small GTPases, including Rac1, Cdc42 and RhoA. Specifically, Rac1, acting in a complex with the MgcRacGAP (male germ cell RacGAP), promotes tyrosine phosphorylation of Stat3 by the IL6-receptor family/Jak kinase complex, as well as its translocation to the nucleus. Studies have further revealed that the mutational activation of Rac1 and Cdc42 results in Stat3 activation, which occurs in part through the upregulation of IL6 family cytokines that in turn stimulates Stat3 through the Jak kinases. Interestingly, evidence also shows that the engagement of cadherins, cell to cell adhesion molecules, specifically induces a striking increase in Rac1 and Cdc42 protein levels and activity, which in turn results in Stat3 activation. In this review we integrate recent findings clarifying the role of the Rho family GTPases in Stat3 activation in the context of malignant progression. © 2011 Elsevier Inc

Stat3 and Gap Junctions in Normal and Lung Cancer Cells

Gap junctions are channels linking the interiors of neighboring cells. A reduction in gap junctional intercellular communication (GJIC) correlates with high cell proliferation, while oncogene products such as Src suppress GJIC, through the Ras/Raf/Erk and other effector pathways. High Src activity was found to correlate with high levels of the Src effector, Signal Transducer and Activator of Transcription-3 (Stat3) in its tyrosine-705 phosphorylated, i.e., transcriptionally activated form, in the majority of Non-Small Cell Lung Cancer lines examined. However, Stat3 inhibition did not restore GJIC in lines with high Src activity. In the contrary, Stat3 inhibition in normal cells or in lines with low Src activity and high GJIC eliminated gap junctional communication. Therefore, despite the fact that Stat3 is growth promoting and in an activated form acts like an oncogene, it is actually required for junctional permeability

Stat3 Is Required for Full Neoplastic Transformation by the Simian Virus 40 Large Tumor Antigen

To investigate the role of Stat3 (signal transducer and activator of transcription-3) in neoplastic transformation by the Large Tumor antigen of Simian Virus 40 (TAg), murine fibroblasts were rendered deficient in Stat3 activity through expression of a Stat3-specific siRNA or a Cre-loxP recombination system. The results demonstrate that growth rate, formation of foci overgrowing a monolayer of normal cells and colony formation in soft agar were dramatically reduced in Stat3-deficient cells. In addition, TAg expression led to increased Stat3 tyrosine phosphorylation, DNA binding, and transcriptional activity, suggesting that Stat3 is required for TAg-mediated neoplasia. Stat3 activation was prevented by blocking the binding of TAg to pRb (retinoblastoma-susceptibility gene product), whereas genetic ablation of pRb increased Stat3 activity, suggesting that pRb inactivation by TAg might be responsible for the observed Stat3 activation. Stat3 activation by TAg was suppressed after inhibition of c-Src, JAKs or the insulin-like growth factor receptor. On the other hand, targeted disruption of the Fer kinase or pharmacological inhibition of Abl had no effect. Inhibition of Src activity led to Stat3 down-regulation as well as apoptosis of sparsely growing, TAg-transformed cells. However, Src inhibition was relatively ineffective in confluent cells, consistent with previous results indicating that cell to cell adhesion activates Stat3 by a Src-independent mechanism. Direct Stat3 inhibition on the other hand induced apoptosis very effectively in confluent cells, which could have significant therapeutic implications. Taken together, our results suggest that Stat3 is an important component of a pathway emanating from TAg and leading to neoplastic conversion

Beyond structure, to survival: activation of Stat3 by cadherin engagement

Cells in normal tissues or in tumors have extensive opportunities for adhesion to their neighbors and the importance of cell to cell contact in the study of fundamental cellular processes in beginning to emerge. In this review, we discuss recent evidence of dramatic changes in the activity of an important signal transducer found to be profoundly affected by cell to cell adhesion, the signal trasducerand activator of transcription-3 (Stat3). Direct cadherin engagement, growth of cells to postconfluence, or formation of multicellular aggregates were found to induce a striking increase in the levels of Stat3 activity, Rac1/Cdc42, and members of the IL6 receptor family in different settings. This activation was specific to Stat3, in that the levels of the extracellular signal regulated kinase (Erk1/2)), a signal transducer often coodinately activated with Stat3 by a number of growth factors or oncogenes, remained unaffected by cell density. Density-dependent Stat3 activation may play a key role in survival, and could contribute to the establishment of cell polarity. It is clear that at any given time the total Stat3 activity levels in a cell are the sum of the effects of cell to cell adhesion plus the conventional Stat3 ac tivating factors present

Stat3 is a positive regulator of gap junctional intercellular communication in cultured, human lung carcinoma cells

Abstract Background Neoplastic transformation of cultured cells by a number of oncogenes such as src suppresses gap junctional, intercellular communication (GJIC); however, the role of Src and its effector Signal transducer and activator of transcription-3 (Stat3) upon GJIC in non small cell lung cancer (NSCLC) has not been defined. Immunohistochemical analysis revealed high Src activity in NSCLC biopsy samples compared to normal tissues. Here we explored the potential effect of Src and Stat3 upon GJIC, by assessing the levels of tyr418-phosphorylated Src and tyr705-phosphorylated Stat3, respectively, in a panel of NSCLC cell lines. Methods Gap junctional communication was examined by electroporating the fluorescent dye Lucifer yellow into cells grown on a transparent electrode, followed by observation of the migration of the dye to the adjacent, non-electroporated cells under fluorescence illumination. Results An inverse relationship between Src activity levels and GJIC was noted; in five lines with high Src activity GJIC was absent, while two lines with extensive GJIC (QU-DB and SK-LuCi6) had low Src levels, similar to a non-transformed, immortalised lung epithelial cell line. Interestingly, examination of the mechanism indicated that Stat3 inhibition in any of the NSCLC lines expressing high endogenous Src activity levels, or in cells where Src was exogenously transduced, did not restore GJIC. On the contrary, Stat3 downregulation in immortalised lung epithelial cells or in the NSCLC lines displaying extensive GJIC actually suppressed junctional permeability. Conclusions Our findings demonstrate that although Stat3 is generally growth promoting and in an activated form it can act as an oncogene, it is actually required for gap junctional communication both in nontransformed lung epithelial cells and in certain lung cancer lines that retain extensive GJIC

Stat3 Activity Is Required for Gap Junctional Permeability in Normal Rat Liver Epithelial Cells

Neoplastic transformation by oncogenes such as activated Src is known to suppress gap junctional, intercellular communication (GJIC). One of the Src effector pathways leading to GJIC suppression and transformation is the Ras/Raf/Mek/Erk, so that inhibition of this pathway in vSrc-transformed cells restores GJIC. A distinct Src downstream effector required for neoplasia is the signal transducer and activator of transcription-3 (Stat3). To examine the role of Stat3 upon the Src-mediated, GJIC suppression, Stat3 was downregulated in rat liver epithelial cells expressing activated Src through treatment with the CPA7, Stat3 inhibitor, or through infection with a retroviral vector expressing a Stat3-specific shRNA. GJIC was examined by electroporating the fluorescent dye, Lucifer yellow, into cells grown on two coplanar electrodes of electrically conductive, optically transparent, indium-tin oxide, followed by observation of the migration of the dye to the adjacent, nonelectroporated cells under fluorescence illumination. The results demonstrate that, contrary to inhibition of the Ras pathway, Stat3 inhibition in cells expressing activated Src does not restore GJIC. On the contrary, Stat3 inhibition in normal cells with high GJIC levels eliminated junctional permeability. Therefore, Stat3's function is actually required for the maintenance of junctional permeability, although Stat3 generally promotes growth and in an activated form can act as an oncogene