RESUMO
Although aberrant activation of the KRAS and PI3K pathway alongside TP53 mutations account for frequent aberrations in human gastric cancers, neither the sequence nor the individual contributions of these mutations have been clarified. Here, we establish an allelic series of mice to afford conditional expression in the glandular epithelium of KrasG12D;Pik3caH1047R or Trp53R172H and/or ablation of Pten or Trp53. We find that KrasG12D;Pik3caH1047R is sufficient to induce adenomas and that lesions progress to carcinoma when also harboring Pten deletions. An additional challenge with either Trp53 loss- or gain-of-function alleles further accelerated tumor progression and triggered metastatic disease. While tumor-intrinsic STAT3 signaling in response to gp130 family cytokines remained as a gatekeeper for all stages of tumor development, metastatic progression required a mutant Trp53-induced interleukin (IL)-11 to IL-6 dependency switch. Consistent with the poorer survival of patients with high IL-6 expression, we identify IL-6/STAT3 signaling as a therapeutic vulnerability for TP53-mutant gastric cancer.
RESUMO
A critical stage of T cell development is ß-selection; at this stage, the T cell receptor ß (TCRß) chain is generated, and the developing T cell starts to acquire antigenic specificity. Progression through ß-selection is assisted by low-affinity interactions between the nascent TCRß chain and peptide presented on stromal major histocompatibility complex and cues provided by the niche. In this study, we identify a cue within the developing T cell niche that is critical for T cell development. E-cadherin mediates cell-cell interactions and influences cell fate in many developmental systems. In developing T cells, E-cadherin contributed to the formation of an immunological synapse and the alignment of the mitotic spindle with the polarity axis during division, which facilitated subsequent T cell development. Collectively, these data suggest that E-cadherin facilitates interactions with the thymic niche to coordinate the ß-selection stage of T cell development.
Assuntos
Caderinas , Linfócitos T , Animais , Camundongos , Caderinas/metabolismo , Comunicação Celular , Receptores de Antígenos de Linfócitos T alfa-beta , Linfócitos T/citologia , Linfócitos T/metabolismo , Timo , Fuso Acromático/metabolismoRESUMO
Aberrant expression of the oncoprotein c-Myc (Myc) is frequently observed in solid tumors and is associated with reduced overall survival. In addition to well-recognized cancer cell-intrinsic roles of Myc, studies have also suggested tumor-promoting roles for Myc in cells of the tumor microenvironment, including macrophages and other myeloid cells. Here, we benchmark Myc inactivation in tumor cells against the contribution of its expression in myeloid cells of murine hosts that harbor endogenous or allograft tumors. Surprisingly, we observe that LysMCre-mediated Myc ablation in host macrophages does not attenuate tumor growth regardless of immunogenicity, the cellular origin of the tumor, the site it develops, or the stage along the tumor progression cascade. Likewise, we find no evidence for Myc ablation to revert or antagonize the polarization of alternatively activated immunosuppressive macrophages. Thus, we surmise that systemic targeting of Myc activity may confer therapeutic benefits primarily through limiting Myc activity in tumor cells rather than reinvigorating the anti-tumor activity of macrophages.
Assuntos
Macrófagos , Neoplasias , Camundongos , Animais , Macrófagos/metabolismo , Neoplasias/metabolismo , Células Mieloides/metabolismo , Microambiente TumoralRESUMO
Multiplexed immunohistochemistry enables analysis of cellular and signaling events in the context of an intact organ. Here, we describe protocols for applying multiplexed immunohistochemistry to the mouse thymus. In particular, we describe how to identify cells at the specific differentiation stage known as ß-selection, and to monitor pre-TCR signaling and the cellular response at that stage. For complete details on the use and execution of this protocol, please refer to Allam et al. (2021).
Assuntos
Ativação Linfocitária , Receptores de Antígenos de Linfócitos T , Animais , Diferenciação Celular , Camundongos , Transdução de Sinais , Linfócitos TRESUMO
Inflammatory breast cancer (IBC) describes a highly aggressive form of breast cancer of diverse molecular subtypes and clonal heterogeneity across individual tumors. Accordingly, IBC is recognized by its clinical signs of inflammation, associated with expression of interleukin (IL)-6 and other inflammatory cytokines. Here, we investigate whether sub-clonal differences between expression of components of the IL-6 signaling cascade reveal a novel role for IL-6 to mediate a proliferative response in trans using two prototypical IBC cell lines. We find that SUM149 and SUM 190 cells faithfully replicate differential expression observed in a subset of human IBC specimens between IL-6, the activated form of the key downstream transcription factor STAT3, and of the HER2 receptor. Surprisingly, the high level of IL-6 produced by SUM149 cells activates STAT3 and stimulates proliferation in SUM190 cells, but not in SUM149 cells with low IL-6R expression. Importantly, SUM149 conditioned medium or co-culture with SUM149 cells induced growth of SUM190 cells, and this effect was abrogated by the IL-6R neutralizing antibody Tocilizumab. The results suggest a novel function for inter-clonal IL-6 signaling in IBC, whereby IL-6 promotes in trans proliferation of IL-6R and HER2-expressing responsive sub-clones and, therefore, may provide a vulnerability that can be exploited therapeutically by repurposing of a clinically approved antibody.
RESUMO
The ß-selection checkpoint of T cell development tests whether the cell has recombined its genomic DNA to produce a functional T cell receptor ß (TCRß). Passage through the ß-selection checkpoint requires the nascent TCRß protein to mediate signaling through a pre-TCR complex. In this study, we show that developing T cells at the ß-selection checkpoint establish an immunological synapse in in vitro and in situ, resembling that of the mature T cell. The immunological synapse is dependent on two key signaling pathways known to be critical for the transition beyond the ß-selection checkpoint, Notch and CXCR4 signaling. In vitro and in situ analyses indicate that the immunological synapse promotes passage through the ß-selection checkpoint. Collectively, these data indicate that developing T cells regulate pre-TCR signaling through the formation of an immunological synapse. This signaling platform integrates cues from Notch, CXCR4, and MHC on the thymic stromal cell to allow transition beyond the ß-selection checkpoint.
Assuntos
Sinapses Imunológicas/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Linfócitos T/imunologia , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Polaridade Celular , Proliferação de Células , Humanos , Complexo Principal de Histocompatibilidade , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Centro Organizador dos Microtúbulos/metabolismo , Modelos Biológicos , Mimetismo Molecular , Receptores CXCR4/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Células Estromais/metabolismo , Linfócitos T/citologia , Timo/metabolismoRESUMO
Cell polarity is an essential process shared by almost all animal tissues. Moreover, cell polarity enables cells to sense and respond to the cues provided by the neighboring cells and the surrounding microenvironment. These responses play a critical role in regulating key physiological processes, including cell migration, proliferation, differentiation, vesicle trafficking and immune responses. The polarity protein complexes regulating these interactions are highly evolutionarily conserved between vertebrates and invertebrates. Interestingly, these polarity complexes interact with each other and key signaling pathways in a cell-polarity context-dependent manner. However, the exact mechanisms by which these interactions take place are poorly understood. In this review, we will focus on the roles of the key polarity complexes SCRIB, PAR and Crumbs in regulating different forms of cell polarity, including epithelial cell polarity, cell migration, asymmetric cell division and the T-cell immunological synapse assembly and signaling.
Assuntos
Polaridade Celular/fisiologia , Microambiente Celular , Animais , Biomarcadores , Movimento Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Humanos , Transdução de SinaisRESUMO
The emerging standard of care for patients with inoperable pancreatic cancer is a combination of cytotoxic drugs gemcitabine and Abraxane, but patient response remains moderate. Pancreatic cancer development and metastasis occur in complex settings, with reciprocal feedback from microenvironmental cues influencing both disease progression and drug response. Little is known about how sequential dual targeting of tumor tissue tension and vasculature before chemotherapy can affect tumor response. We used intravital imaging to assess how transient manipulation of the tumor tissue, or "priming," using the pharmaceutical Rho kinase inhibitor Fasudil affects response to chemotherapy. Intravital Förster resonance energy transfer imaging of a cyclin-dependent kinase 1 biosensor to monitor the efficacy of cytotoxic drugs revealed that priming improves pancreatic cancer response to gemcitabine/Abraxane at both primary and secondary sites. Transient priming also sensitized cells to shear stress and impaired colonization efficiency and fibrotic niche remodeling within the liver, three important features of cancer spread. Last, we demonstrate a graded response to priming in stratified patient-derived tumors, indicating that fine-tuned tissue manipulation before chemotherapy may offer opportunities in both primary and metastatic targeting of pancreatic cancer.
Assuntos
Progressão da Doença , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Quinases Associadas a rho/antagonistas & inibidores , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/uso terapêutico , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Paclitaxel Ligado a Albumina/farmacologia , Paclitaxel Ligado a Albumina/uso terapêutico , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Técnicas Biossensoriais , Proteína Quinase CDC2/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Colágeno/metabolismo , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Matriz Extracelular/metabolismo , Humanos , Fígado/patologia , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Transdução de Sinais/efeitos dos fármacos , Resultado do Tratamento , Quinases Associadas a rho/metabolismo , Quinases da Família src/metabolismo , GencitabinaRESUMO
ROCK signaling causes epidermal hyper-proliferation by increasing ECM production, elevating dermal stiffness, and enhancing Fak-mediated mechano-transduction signaling. Elevated dermal stiffness in turn causes ROCK activation, establishing mechano-reciprocity, a positive feedback loop that can promote tumors. We have identified a negative feedback mechanism that limits excessive ROCK signaling during wound healing and is lost in squamous cell carcinomas (SCCs). Signal flux through ROCK was selectively tuned down by increased levels of 14-3-3ζ, which interacted with Mypt1, a ROCK signaling antagonist. In 14-3-3ζ(-/-) mice, unrestrained ROCK signaling at wound margins elevated ECM production and reduced ECM remodeling, increasing dermal stiffness and causing rapid wound healing. Conversely, 14-3-3ζ deficiency enhanced cutaneous SCC size. Significantly, inhibiting 14-3-3ζ with a novel pharmacological agent accelerated wound healing 2-fold. Patient samples of chronic non-healing wounds overexpressed 14-3-3ζ, while cutaneous SCCs had reduced 14-3-3ζ. These results reveal a novel 14-3-3ζ-dependent mechanism that negatively regulates mechano-reciprocity, suggesting new therapeutic opportunities.
Assuntos
Proteínas 14-3-3/metabolismo , Proliferação de Células/fisiologia , Homeostase/fisiologia , Transdução de Sinais/fisiologia , Cicatrização/fisiologia , Quinases Associadas a rho/metabolismo , Animais , Epiderme/metabolismo , CamundongosRESUMO
INTRODUCTION: Mammographic density (MD), after adjustment for a women's age and body mass index, is a strong and independent risk factor for breast cancer (BC). Although the BC risk attributable to increased MD is significant in healthy women, the biological basis of high mammographic density (HMD) causation and how it raises BC risk remain elusive. We assessed the histological and immunohistochemical differences between matched HMD and low mammographic density (LMD) breast tissues from healthy women to define which cell features may mediate the increased MD and MD-associated BC risk. METHODS: Tissues were obtained between 2008 and 2013 from 41 women undergoing prophylactic mastectomy because of their high BC risk profile. Tissue slices resected from the mastectomy specimens were X-rayed, then HMD and LMD regions were dissected based on radiological appearance. The histological composition, aromatase immunoreactivity, hormone receptor status and proliferation status were assessed, as were collagen amount and orientation, epithelial subsets and immune cell status. RESULTS: HMD tissue had a significantly greater proportion of stroma, collagen and epithelium, as well as less fat, than LMD tissue did. Second harmonic generation imaging demonstrated more organised stromal collagen in HMD tissues than in LMD tissues. There was significantly more aromatase immunoreactivity in both the stromal and glandular regions of HMD tissues than in those regions of LMD tissues, although no significant differences in levels of oestrogen receptor, progesterone receptor or Ki-67 expression were detected. The number of macrophages within the epithelium or stroma did not change; however, HMD stroma exhibited less CD206(+) alternatively activated macrophages. Epithelial cell maturation was not altered in HMD samples, and no evidence of epithelial-mesenchymal transition was seen; however, there was a significant increase in vimentin(+)/CD45(+) immune cells within the epithelial layer in HMD tissues. CONCLUSIONS: We confirmed increased proportions of stroma and epithelium, increased aromatase activity and no changes in hormone receptor or Ki-67 marker status in HMD tissue. The HMD region showed increased collagen deposition and organisation as well as decreased alternatively activated macrophages in the stroma. The HMD epithelium may be a site for local inflammation, as we observed a significant increase in CD45(+)/vimentin(+) immune cells in this area.