Fis1 phosphorylation by Met promotes mitochondrial fission and hepatocellular carcinoma metastasis.

Met tyrosine kinase, a receptor for a hepatocyte growth factor (HGF), plays a critical role in tumor growth, metastasis, and drug resistance. Mitochondria are highly dynamic and undergo fission and fusion to maintain a functional mitochondrial network. Dysregulated mitochondrial dynamics are responsible for the progression and metastasis of many cancers. Here, using structured illumination microscopy (SIM) and high spatial and temporal resolution live cell imaging, we identified mitochondrial trafficking of receptor tyrosine kinase Met. The contacts between activated Met kinase and mitochondria formed dramatically, and an intact HGF/Met axis was necessary for dysregulated mitochondrial fission and cancer cell movements. Mechanically, we found that Met directly phosphorylated outer mitochondrial membrane protein Fis1 at Tyr38 (Fis1 pY38). Fis1 pY38 promoted mitochondrial fission by recruiting the mitochondrial fission GTPase dynamin-related protein-1 (Drp1) to mitochondria. Fragmented mitochondria fueled actin filament remodeling and lamellipodia or invadopodia formation to facilitate cell metastasis in hepatocellular carcinoma (HCC) cells both in vitro and in vivo. These findings reveal a novel and noncanonical pathway of Met receptor tyrosine kinase in the regulation of mitochondrial activities, which may provide a therapeutic target for metastatic HCC.

FUT8-mediated aberrant N-glycosylation of B7H3 suppresses the immune response in triple-negative breast cancer.

Most patients with triple negative breast cancer (TNBC) do not respond to anti-PD1/PDL1 immunotherapy, indicating the necessity to explore immune checkpoint targets. B7H3 is a highly glycosylated protein. However, the mechanisms of B7H3 glycosylation regulation and whether the sugar moiety contributes to immunosuppression are unclear. Here, we identify aberrant B7H3 glycosylation and show that N-glycosylation of B7H3 at NXT motif sites is responsible for its protein stability and immunosuppression in TNBC tumors. The fucosyltransferase FUT8 catalyzes B7H3 core fucosylation at N-glycans to maintain its high expression. Knockdown of FUT8 rescues glycosylated B7H3-mediated immunosuppressive function in TNBC cells. Abnormal B7H3 glycosylation mediated by FUT8 overexpression can be physiologically important and clinically relevant in patients with TNBC. Notably, the combination of core fucosylation inhibitor 2F-Fuc and anti-PDL1 results in enhanced therapeutic efficacy in B7H3-positive TNBC tumors. These findings suggest that targeting the FUT8-B7H3 axis might be a promising strategy for improving anti-tumor immune responses in patients with TNBC.

Disruption of super-enhancer-driven tumor suppressor gene RCAN1.4 expression promotes the malignancy of breast carcinoma.

BACKGROUND: Super-enhancers (SEs) play a crucial role in cancer, which is often associate with activated oncogenes. However, little is known about how SEs facilitate tumour suppression. Individuals with Down syndrome exhibit a remarkably reduced incidence of breast cancer (BC), moving the search for tumor suppressor genes on human chromosome 21 (HSA21). In this study, we aim to identify and explore potential mechanisms by which SEs are established for tumor suppressor RCAN1.4 on HSA21 in BC. METHODS: In silico analysis and immunohistochemical staining were used to assess the expression and clinical relevance of RCAN1.4 and RUNX3 in BC. Function experiments were performed to evaluate the effects of RCAN1.4 on the malignancy of breast carcinoma in vitro and in vivo. ChIP-seq data analysis, ChIP-qPCR, double-CRISPR genome editing, and luciferase reporter assay were utilized to confirm RUNX3 was involved in regulating RCAN1.4-associated SE in BC. The clinical value of co-expression of RCAN1.4 and RUNX3 was evaluated in BC patients. RESULTS: Here, we characterized RCAN1.4 as a potential tumour suppressor in BC. RCAN1.4 loss promoted tumour metastasis to bone and brain, and its overexpression inhibited tumour growth by blocking the calcineurin-NFATc1 pathway. Unexpectedly, we found RCAN1.4 expression was driven by a ~ 23 kb-long SE. RCAN1.4-SEdistal was sensitive to BRD4 inhibition, and its deletion decreased RCAN1.4 expression by over 90% and induced the malignant phenotype of BC cells. We also discovered that the binding sites in the SE region of RCAN1.4 were enriched for consensus sequences of transcription factor RUNX3. Knockdown of RUNX3 repressed the luciferase activity and also decreased H3K27ac enrichment binding at the SE region of RCAN1.4. Furthermore, abnormal SE-driven RCAN1.4 expression mediated by RUNX3 loss could be physiologically significant and clinically relevant in BC patients. Notably, we established a prognostic model based on RCAN1.4 and RUNX3 co-expression that effectively predicted the overall survival in BC patients. CONCLUSIONS: These findings reveal an important role of SEs in facilitating tumour suppression in BC. Considering that the combination of low RCAN1.4 and low RUNX3 expression has worse prognosis, RUNX3-RCAN1.4 axis maybe a novel prognostic biomarker and therapeutic target for BC patients.

Autophagy deficiency promotes triple-negative breast cancer resistance to T cell-mediated cytotoxicity by blocking tenascin-C degradation.

Most triple-negative breast cancer (TNBC) patients fail to respond to T cell-mediated immunotherapies. Unfortunately, the molecular determinants are still poorly understood. Breast cancer is the disease genetically linked to a deficiency in autophagy. Here, we show that autophagy defects in TNBC cells inhibit T cell-mediated tumour killing in vitro and in vivo. Mechanistically, we identify Tenascin-C as a candidate for autophagy deficiency-mediated immunosuppression, in which Tenascin-C is Lys63-ubiquitinated by Skp2, particularly at Lys942 and Lys1882, thus promoting its recognition by p62 and leading to its selective autophagic degradation. High Tenascin-C expression is associated with poor prognosis and inversely correlated with LC3B expression and CD8+ T cells in TNBC patients. More importantly, inhibition of Tenascin-C in autophagy-impaired TNBC cells sensitizes T cell-mediated tumour killing and improves antitumour effects of single anti-PD1/PDL1 therapy. Our results provide a potential strategy for targeting TNBC with the combination of Tenascin-C blockade and immune checkpoint inhibitors.

Polo-Like Kinase 1 phosphorylates and stabilizes KLF4 to promote tumorigenesis in nasopharyngeal carcinoma.

Rationale: Advanced nasopharyngeal carcinoma (NPC) is an aggressive disease with no targeted therapies and poor outcomes. New innovative targets are urgently needed. KLF4 has been extensively studied in the context of tumors, and current data suggest that it can act as either a tissue-specific tumor-inhibiting or a tumor-promoting gene. Here, we found that KLF4 played as a tumor-promoting gene in NPC, and could be mediated by PLK1. Methods: Tissue immunohistochemistry (IHC) assay was performed to identify the role of KLF4 in NPC. Global gene expression experiments were performed to explore the molecular mechanisms underlying KLF4-dependent tumorigenesis. Small-molecule kinase inhibitor screening was performed to identify potential upstream kinases of KLF4. The pharmacologic activity of polo-like kinase inhibitor volasertib (BI6727) in vitro and in vivo was determined. Result: Our investigation showed that high expression of KLF4 was correlated with poor prognosis in NPC. Moreover, genome-wide profiling revealed that KLF4 directly activated oncogenic programmes, including gene sets associated with KRAS, VEGF, and MYC signalling. We further found that inhibition of polo-like kinase 1 could downregulate the expression of KLF4 and that PLK1 directly phosphorylated KLF4 at Ser234. Notably, phosphorylation of KLF4 by PLK1 caused the recruitment and binding of the E3 ligase TRAF6, which resulted in KLF4 K32 K63-linked ubiquitination and stabilization. Moreover, KLF4 could enhance TRAF6 expression at the transcriptional level, thus initiating a KLF4-TRAF6 feed-forward loop. Treatment with the PLK1 inhibitor volasertib (BI6727) significantly inhibited tumor growth in nude mice. Conclusion: Our study unveiled a new PLK1-TRAF6-KLF4 feed-forward loop. The resulting increase in KLF4 ubiquitination leads to stabilization and upregulation of KLF4, which leads to tumorigenesis in NPC. These results expand our understanding of the role of KLF4 in NPC and validate PLK1 inhibitors as potential therapeutic agents for NPC, especially cancer patients with KLF4 overexpression.

Regorafenib Promotes Antitumor Immunity via Inhibiting PD-L1 and IDO1 Expression in Melanoma.

PURPOSE: Immune checkpoint blockade (ICB) therapy induces durable tumor regressions in a minority of patients with cancer. In this study, we aimed to identify kinase inhibitors that were capable of increasing the antimelanoma immunity. EXPERIMENTAL DESIGN: Flow cytometry-based screening was performed to identify kinase inhibitors that can block the IFNγ-induced PD-L1 expression in melanoma cells. The pharmacologic activities of regorafenib alone or in combination with immunotherapy in vitro and in vivo were determined. The mechanisms of regorafenib were explored and analyzed in melanoma patients treated with or without anti-PD-1 using The Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) datasets. RESULTS: Through screening of a kinase inhibitor library, we found approximately 20 agents that caused more than half reduction of cell surface PD-L1 level, and regorafenib was one of the most potent agents. Furthermore, our results showed that regorafenib, in vitro and in vivo, strongly promoted the antitumor efficacy when combined with IFNγ or ICB. By targeting the RET-Src axis, regorafenib potently inhibited JAK1/2-STAT1 and MAPK signaling and subsequently attenuated the IFNγ-induced PD-L1 and IDO1 expression without affecting MHC-I expression much. Moreover, RET and Src co-high expression was an independent unfavorable prognosis factor in melanoma patients with or without ICB through inhibiting the antitumor immune response. CONCLUSIONS: Our data unveiled a new mechanism of alleviating IFNγ-induced PD-L1 and IDO1 expression and provided a rationale to explore a novel combination of ICB with regorafenib clinically, especially in melanoma with RET/Src axis activation.