RESUMEN
How basal cell carcinoma (BCC) interacts with its tumor microenvironment to promote growth is unclear. We use singe-cell RNA sequencing to define the human BCC ecosystem and discriminate between normal and malignant epithelial cells. We identify spatial biomarkers of tumors and their surrounding stroma that reinforce the heterogeneity of each tissue type. Combining pseudotime, RNA velocity-PAGA, cellular entropy, and regulon analysis in stromal cells reveals a cancer-specific rewiring of fibroblasts, where STAT1, TGF-ß, and inflammatory signals induce a noncanonical WNT5A program that maintains the stromal inflammatory state. Cell-cell communication modeling suggests that tumors respond to the sudden burst of fibroblast-specific inflammatory signaling pathways by producing heat shock proteins, whose expression we validated in situ. Last, dose-dependent treatment with an HSP70 inhibitor suppresses in vitro vismodegib-resistant BCC cell growth, Hedgehog signaling, and in vivo tumor growth in a BCC mouse model, validating HSP70's essential role in tumor growth and reinforcing the critical nature of tumor microenvironment cross-talk in BCC progression.
Asunto(s)
Carcinoma Basocelular , Neoplasias Cutáneas , Animales , Carcinoma Basocelular/tratamiento farmacológico , Carcinoma Basocelular/genética , Carcinoma Basocelular/metabolismo , Ecosistema , Proteínas Hedgehog , Humanos , Ratones , Análisis de la Célula Individual , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Microambiente TumoralRESUMEN
Basal cell carcinoma (BCC) is a locally invasive epithelial cancer that is primarily driven by the Hedgehog (HH) pathway. Advanced BCCs are a critical subset of BCCs that frequently acquire resistance to Smoothened (SMO) inhibitors and identifying pathways that bypass SMO could provide alternative treatments for patients with advanced or metastatic BCC. Here, we use a combination of RNA-sequencing analysis of advanced human BCC tumor-normal pairs and immunostaining of human and mouse BCC samples to identify a PI3K pathway expression signature in BCC. Pharmacological inhibition of PI3K activity in BCC cells significantly reduces cell proliferation and HH signaling. However, treatment of Ptch1fl/fl ; Gli1-CreERT2 mouse BCCs with the PI3K inhibitor BKM120 results in a reduction of tumor cell growth with no significant effect on HH signaling. Downstream PI3K components aPKC and Akt1 showed a reduction in active protein, whereas their substrate, cyclin-dependent kinase inhibitor p21, showed a concomitant increase in protein stability. Our results suggest that PI3K promotes BCC tumor growth by kinase-induced p21 degradation without altering HH signaling.
RESUMEN
Advanced basal cell carcinomas (BCCs) are driven by the Hedgehog (HH) pathway and often possess inherent resistance to SMO inhibitors. Identifying and targeting pathways that bypass SMO could provide alternative treatments for patients with advanced or metastatic BCC. Here, we use a combination of RNA-sequencing analysis of advanced human BCC tumor-normal pairs and immunostaining of human and mouse BCC samples to identify an MTOR expression signature in BCC. Pharmacological inhibition of MTOR activity in BCC cells significantly reduces cell proliferation without affecting HH signalling. Similarly, treatment of the Ptch1 fl/fl ; Gli1-CreERT2 mouse BCC tumor model with everolimus reduces tumor growth. aPKC, a downstream target of MTOR, shows reduced activity, suggesting that MTOR promotes tumor growth by activating aPKC and demonstrating that suppressing MTOR could be a promising target for BCC patients.
Asunto(s)
Carcinoma Basocelular/genética , Carcinoma Basocelular/metabolismo , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Serina-Treonina Quinasas TOR , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Carcinoma Basocelular/tratamiento farmacológico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Everolimus/farmacología , Everolimus/uso terapéutico , Proteínas Hedgehog/metabolismo , Humanos , Imidazoles/farmacología , Inmunohistoquímica , Ratones , Receptor Patched-1/genética , Proteína Quinasa C/metabolismo , Análisis de Secuencia de ARN , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Neoplasias Cutáneas/tratamiento farmacológico , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Triazinas/farmacología , Proteína con Dedos de Zinc GLI1/genéticaRESUMEN
In RAW 264.7 cells, PKC-ε regulates FcγR-mediated phagocytosis. BMDM behave similarly; PKC-ε concentrates at phagosomes and internalization are reduced in PKC-εâ»/â» cells. Two questions were asked: what is the role of PKC-ε? and what domains are necessary for PKC-ε concentration? Function was studied using BMDM and frustrated phagocytosis. On IgG surfaces, PKC-εâ»/â» macrophages spread less than WT. Patch-clamping revealed that the spreading defect is a result of the failure of PKC-εâ»/â» macrophages to add membrane. The defect is specific for FcγR ligation and can be reversed by expression of full-length (but not the isolated RD) PKC-ε in PKC-εâ»/â» BMDM. Thus, PKC-ε function in phagocytosis requires translocation to phagosomes and the catalytic domain. The expression of chimeric PKC molecules in RAW cells identified the εPS as necessary for PKC-ε targeting. When placed into (nonlocalizing) PKC-δ, εPS was sufficient for concentration, albeit to a lesser degree than intact PKC-ε. In contrast, translocation of δ(εPSC1B) resembled that of WT PKC-ε. Thus, εPS and εC1B cooperate for optimal phagosome targeting. Finally, cells expressing εK437W were significantly less phagocytic than their PKC-ε-expressing counterparts, blocked at the pseudopod-extension phase. In summary, we have shown that εPS and εC1B are necessary and sufficient for targeting PKC-ε to phagosomes, where its catalytic activity is required for membrane delivery and pseudopod extension.