Cisplatin resistance is a major challenge for advanced head and neck cancer (HNC). Understanding the underlying mechanisms and developing effective strategies against cisplatin resistance are highly desired in the clinic. However, how tumor stroma modulates HNC growth and chemoresistance is unclear. We show that cancer-associated fibroblasts (CAFs) are intrinsically resistant to cisplatin and have an active role in regulating HNC cell survival and proliferation by delivering functional miR-196a from CAFs to tumor cells via exosomes. Exosomal miR-196a then binds novel targets, CDKN1B and ING5, to endow HNC cells with cisplatin resistance. Exosome or exosomal miR-196a depletion from CAFs functionally restored HNC cisplatin sensitivity. Importantly, we found that miR-196a packaging into CAF-derived exosomes might be mediated by heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1). Moreover, we also found that high levels of plasma exosomal miR-196a are clinically correlated with poor overall survival and chemoresistance. The present study finds that CAF-derived exosomal miR-196a confers cisplatin resistance in HNC by targeting CDKN1B and ING5, indicating miR-196a may serve as a promising predictor of and potential therapeutic target for cisplatin resistance in HNC.

Although immunotherapy can prolong survival in some patients with head and neck squamous cell carcinoma (HNSCC), the response rate remains low. Clarification of the critical mechanisms regulating CD8+ T-cell infiltration and dysfunction in the tumor microenvironment could help maximize the benefit of immunotherapy for treating HNSCC. Here, we performed spatial transcriptomic analysis of HNSCC specimens with differing immune infiltration and single-cell RNA sequencing of five pairs of tumor and adjacent tissues, revealing specific cancer-associated fibroblast (CAF) subsets related to CD8+ T-cell infiltration restriction and dysfunction. These CAFs exhibited high expression of CXCLs (CXCL9, CXCL10, and CXCL12) and MHC-I and enrichment of galectin-9 (Gal9). The proportion of MHC-IhiGal9+ CAFs was inversely correlated with abundance of a TCF1+GZMK+ subset of CD8+ T cells. Gal9 on CAFs induced CD8+ T-cell dysfunction and decreased the proportion of tumor-infiltrating TCF1+CD8+ T cells. Collectively, the identification of MHC-IhiGal9+ CAFs advances the understanding of the precise role of CAFs in cancer immune evasion and paves the way for more effective immunotherapy for HNSCC. Significance: Spatial analysis identifies IFN-induced MHC-IhiGal9+ CAFs that form a trap for CD8+ T cells, providing insights into the complex networks in the tumor microenvironment that regulate T-cell infiltration and function.

Background: Head and neck squamous cell carcinoma (HNSCC) is a very aggressive disease characterized by a heterogeneous tumor immune microenvironment (TIME). Tumor-associated macrophages (TAMs) constitute the major innate immune population in the TIME where they facilitate crucial regulatory processes that participate in malignant tumor progression. SPP1 + macrophages (SPP1 + Macs) are found in many cancers, but their effects on HNSCC remain unknown. This study aimed to identify and validate the role and function of SPP1 + Macs in the malignant progression of HNSCC. Methods: In this study, we applied single-cell RNA sequencing (scRNA-seq) analyses of paired tumor and normal tissues from 5 HNSCC patients to identify tumor-specific SPP1 + Macs. RT-qPCR and multiplex immunohistochemical and multiplex immunofluorescence staining were used to verify the presence of SPP1 + Macs in the clinical samples. Gene set variation analysis suggested that SPP1 + Macs were actively involved in cytokine production. Thus, we constructed SPP1-OE macrophages and SPP1-KD macrophages (both differentiated from THP-1 cells), performed a Luminex liquid suspension chip detection assay to detect differential cytokines, and further assessed their biological functions and mechanisms in several HNSCC cell lines and adjacent macrophages. An in vivo experiment was used to verify the function of SPP1 + Macs in HNSCC progression. Results: The scRNA-seq results revealed that myeloid cells were heterogeneous and strongly correlated with tumor cells in the TIME in HNSCC and identified tumor-specific SPP1 + Macs, which were positively correlated with poor prognosis of HNSCC patients. Gene set variation analysis (GSVA) suggested that SPP1 + Macs were actively involved in cytokine production. Luminex liquid suspension chip detection assay indicated that SPP1 + Mac-derived TNF-α and IL-1β played important roles. Both in vitro and in vivo experiments and the use of VGX-1027, an inhibitor of macrophage-derived TNF-α and IL-1β, confirmed that SPP1 + Mac-derived TNF-α and IL-1β promoted HNSCC progression by supporting tumor cell proliferation and migration. Mechanistically, we found that TNF-α and IL-1β were upregulated due to NF-kappa B signaling pathway activation in SPP1 + Macs. Moreover, SPP1 + Mac-derived TNF-α and IL-1β promoted the expression of OPN in both tumor cells and other adjacent macrophages through different signaling pathways. Conclusions: SPP1 + Macs increase the secretion of TNF-α and IL-1β via the NF-kappa B pathway to promote HNSCC cell proliferation, and TNF-α and IL-1β in turn upregulate the expression of OPN in tumor cells and macrophages; thus, SPP1 + Macs may be a candidate target through which antitumor efficacy can be enhanced.

The Toll-like receptor 2 (TLR2)-driven tissue response may promote neoangiogenesis and tumour growth by mechanisms that are poorly understood. We investigated the expression levels of TLR2 and associated-miRNAs in colorectal carcinoma (CRC) tissues and cell lines using real-time PCR, northern blotting and western blotting. Survival curver was generated by Log-Rank test and the role of TLR2 signalling in tumour invasion and migration was determined by transwell analysis kits. We observed that the tissues from CRC patients express relatively high levels of TLR2. Targeting TLR2 markedly reduces the invasion and migration of CRC cells. We also found that miR-143, a putative tumour suppressor that is down-regulated in CRC tissues, reduces the invasion and migration of CRC cells primarily via TLR2. Utilising a xenograft mouse model, we demonstrated that re-expression of miR-143 inhibits CRC cell colonisation in vivo. miR-143 blocks the TLR2 signalling pathway in human CRC cells. This knowledge may pave the way for new clinical applications utilising miR-143 mimics in the treatment of patients with CRC.

Although the chemopreventive effects of aspirin have been extensively investigated, the roles of many cell components, such as long non-coding RNAs, in these effects are still not completely understood. We identify an aspirin-induced upregulated lncRNA, OLA1P2, in human colorectal cancer. Aspirin induces demethylation of the FOXD3 promoter and promotes expression of the FOXD3 gene. Subsequently, upregulated FOXD3 protein transcriptionally activates lncRNA OLA1P2 expression. OLA1P2 upregulation markedly affects STAT3 signaling pathway activity by inhibiting the nuclear import of phosphorylated STAT3. The phosphorylation of tyrosine-705 of STAT3 is the first step in OLA1P2 binding, and the formation of phosphorylated STAT3 homodimers is subsequently blocked. OLA1P2 interacts directly with STAT3 due to OLA1P2 sharing the same conservative STAT3 transcription response element as STAT3 targets. Regular use of aspirin dramatically decreases the number of metastatic nodules of cancer cells in immunodeficient mouse lungs, and OLA1P2 silencing markedly weakens the anti-metastatic activity of aspirin in the lungs. Additionally, low OLA1P2 levels are associated with malignant transformation and lower overall survival in cancers. The present study finds that the aspirin-FOXD3-OLA1P2-STAT3 axis exhibits exciting anticancer effects and provides new insights into the chemopreventive mechanisms underlying aspirin use.

Background: MicroRNA-145 (miR-145) is considered to play key roles in many cellular processes, such as proliferation, differentiation and apoptosis, by inhibiting target gene expression. DNA Fragmentation Factor-45 (DFF45) has been found to be the substrate of Caspase-3, and the cleavage of DFF45 by caspase-3 during apoptosis releases DFF40 that degrades chromosomal DNA into nucleosomal fragments. There are currently no in-depth studies on the relationship between miR-145 and the DFF45 gene.Results: In this study, we identified DFF45 as a novel target of miR-145. We demonstrated that miR-145 targets a putative binding site in the coding sequence (CDS) of DFF45, and its abundance is inversely associated with DFF45 expression in colon cancer cells. Using a luciferase reporter system, we found that miR-145 suppresses the expression of the luciferase reporter gene fused to the putative binding site of DFF45. The level of DFF45 protein, but not DFF45 mRNA, was decreased by miR-145, suggesting a mechanism of translational regulation. Furthermore, we demonstrate that this specific silencing of DFF45 by miR-145 accounts, at least in part, for the staurosporine-induced tumor cell apoptosisin vitro.Conclusions: Our study reveals a previously unrecognized function of miR-145 in DFF45 processing, which may underlie crucial aspects of cancer biology.