PolyI:C attenuates transforming growth factor‐β signaling to induce cytostasis of surrounding cells by secreted factors in triple‐negative breast cancer

The activation of RIG‐I‐like receptor (RLR) signaling in cancer cells is widely recognized as a critical cancer therapy method. The expected mechanism of RLR ligand‐mediated cancer therapy involves the promotion of cancer cell death and strong induction of interferon (IFN)‐β that affects the tumor microenvironment. We have recently shown that activation of RLR signaling in triple‐negative breast cancer cells (TNBC) attenuates transforming growth factor‐β (TGF‐β) signaling, which partly contributes to the promotion of cancer cell pyroptosis. However, the consequences of suppression of TGF‐β signaling by RLR ligands with respect to IFN‐β‐mediated tumor suppression are not well characterized. This study showed that transfection of a typical RLR ligand polyI:C in cancer cells produces significant levels of IFN‐β, which inhibits the growth of the surrounding cancer cells. In addition, IFN‐β‐induced cell cycle arrest in surrounding cancer cells was inhibited by the expression of constitutively active Smad3. Constitutively active Smad3 suppresses IFN‐β expression through the alleviation of IFN regulatory factor 3 binding to the canonical target genes, as suggested by ChIP sequencing analysis. Based on these findings, a new facet of the protumorigenic function of TGF‐β that suppresses IFN‐β expression is suggested when RLR‐mediated cancer treatment is used in TNBC.     

Dual inhibition of TGF‐β and PD‐L1: a novel approach to cancer treatment

Transforming growth factor‐β (TGF‐β) and programmed death ligand 1 (PD‐L1) initiate signaling pathways with complementary, nonredundant immunosuppressive functions in the tumor microenvironment (TME). In the TME, dysregulated TGF‐β signaling suppresses antitumor immunity and promotes cancer fibrosis, epithelial‐to‐mesenchymal transition, and angiogenesis. Meanwhile, PD‐L1 expression inactivates cytotoxic T cells and restricts immunosurveillance in the TME. Anti‐PD‐L1 therapies have been approved for the treatment of various cancers, but TGF‐β signaling in the TME is associated with resistance to these therapies. In this review, we discuss the importance of the TGF‐β and PD‐L1 pathways in cancer, as well as clinical strategies using combination therapies that block these pathways separately or approaches with dual‐targeting agents (bispecific and bifunctional immunotherapies) that may block them simultaneously. Currently, the furthest developed dual‐targeting agent is bintrafusp alfa. This drug is a first‐in‐class bifunctional fusion protein that consists of the extracellular domain of the TGF‐βRII receptor (a TGF‐β ‘trap’) fused to a human immunoglobulin G1 (IgG1) monoclonal antibody blocking PD‐L1. Given the immunosuppressive effects of the TGF‐β and PD‐L1 pathways within the TME, colocalized and simultaneous inhibition of these pathways may potentially improve clinical activity and reduce toxicity.     

Improving function of cytotoxic T‐lymphocytes by transforming growth factor‐β inhibitor in oral squamous cell carcinoma

Immunotherapy with immune‐checkpoint therapy has recently been used to treat oral squamous cell carcinomas (OSCCs). However, improvements in current immunotherapy are expected because response rates are limited. Transforming growth factor‐β (TGF‐β) creates an immunosuppressive tumor microenvironment (TME) by inducing the production of regulatory T‐cells (Tregs) and cancer‐associated fibroblasts and inhibiting the function of cytotoxic T‐lymphocytes (CTLs) and natural killer cells. TGF‐β may be an important target in the development of novel cancer immunotherapies. In this study, we investigated the suppressive effect of TGF‐β on CTL function in vitro using OSCC cell lines and their specific CTLs. Moreover, TGFB1 mRNA expression and T‐cell infiltration in 25 OSCC tissues were examined by in situ hybridization and multifluorescence immunohistochemistry. We found that TGF‐β suppressed the function of antigen‐specific CTLs in the priming and effector phases in vitro. Additionally, TGF‐β inhibitor effectively restored the CTL function, and TGFB1 mRNA was primarily expressed in the tumor invasive front. Interestingly, we found a significant negative correlation between TGFB1 mRNA expression and the CD8⁺ T‐cell/Treg ratio and between TGFB1 mRNA expression and the Ki‐67 expression in CD8⁺ T‐cells, indicating that TGF‐β also suppressed the function of CTLs in situ. Our findings suggest that the regulation of TGF‐β function restores the immunosuppressive TME to active status and is important for developing new immunotherapeutic strategies, such as a combination of immune‐checkpoint inhibitors and TGF‐β inhibitors, for OSCCs.     

BMP9‐ID1 signaling promotes EpCAM‐positive cancer stem cell properties in hepatocellular carcinoma

The malignant nature of hepatocellular carcinoma (HCC) is closely related to the presence of cancer stem cells (CSCs). Bone morphologic protein 9 (BMP9), a member of the transforming growth factor‐beta (TGF‐β) superfamily, was recently reported to be involved in liver diseases including cancer. We aimed to elucidate the role of BMP9 signaling in HCC‐CSC properties and to assess the therapeutic effect of BMP receptor inhibitors in HCC. We have identified that high BMP9 expression in tumor tissues or serum from patients with HCC leads to poorer outcome. BMP9 promoted CSC properties in epithelial cell adhesion molecule (EpCAM)‐positive HCC subtype via enhancing inhibitor of DNA‐binding protein 1 (ID1) expression in vitro. Additionally, ID1 knockdown significantly repressed BMP9‐promoted HCC‐CSC properties by suppressing Wnt/β‐catenin signaling. Interestingly, cells treated with BMP receptor inhibitors {"type":"entrez-nucleotide","attrs":{"text":"K02288","term_id":"191391"}}K02288 and LDN‐212854 blocked HCC‐CSC activation by inhibiting BMP9‐ID1 signaling, in contrast to cells treated with the TGF‐β receptor inhibitor galunisertib. Treatment with LDN‐212854 suppressed HCC tumor growth by repressing ID1 and EpCAM in vivo. Our study demonstrates the pivotal role of BMP9‐ID1 signaling in promoting HCC‐CSC properties and the therapeutic potential of BMP receptor inhibitors in treating EpCAM‐positive HCC. Therefore, targeting BMP9‐ID1 signaling could offer novel therapeutic options for patients with malignant HCC.     

HPV+ HNSCC‐derived exosomal miR‐9‐5p inhibits TGF‐β signaling‐mediated fibroblast phenotypic transformation through NOX4

Human papillomavirus (HPV) is a significant risk factor for head and neck squamous cell carcinoma (HNSCC). HPV⁺ HNSCC patients have a higher survival rate, which may be related to its unique tumor microenvironment. Exosomes are emerging as a communication tool between tumor cells and the tumor microenvironment, including cancer‐associated fibroblasts (CAFs). In this study, 111 clinical samples tissues and public sequencing data were analyzed. Our study found fewer CAFs infiltrated in HPV⁺ HNSCC, and poor CAF infiltration level was associated with a good prognosis. HPV⁺ HNSCC cell‐derived exosomes can significantly reduce the phenotypic transformation of fibroblasts. miR‐9‐5p, as a miRNA enriched in HPV⁺ HNSCC cell‐derived exosomes, can be transferred to fibroblasts. miR‐9‐5p mimic transfection decreased the expression of NOX4 and the level of intracellular reactive oxygen species (ROS), which inhibited the transforming growth factor beta 1(TGF‐β1)‐induced increase of αSMA levels. Therefore, these results indicated that HPV⁺ HNSCC‐derived exosomal miR‐9‐5p inhibits TGF‐β signaling‐mediated fibroblast phenotypic transformation through NOX4, which is related to the excellent prognosis of HPV patients.     

Small extracellular vesicles deliver TGF‐β1 and promote adriamycin resistance in breast cancer cells

Chemotherapeutic resistance is a major obstacle in the control of advanced breast cancer (BCa). We have previously shown that small extracellular vesicles (sEVs) can transmit adriamycin resistance between BCa cells. Here, we describe that sEV‐mediated TGF‐β1 intercellular transfer is involved in the drug‐resistant transmission. sEVs were isolated and characterized from both sensitive and resistant cells. sEVs derived from the resistant cells were incubated with the sensitive cells and resulted in transmitting the resistant phenotype to the recipient cells. Cytokine antibody microarray revealed that most metastasis‐associated cytokines present at the high levels in sEVs from the resistant cells compared with their levels in sEVs from the sensitive cells, particularly TGF‐β1 is enriched in sEVs from the resistant cells. The sEV‐mediated TGF‐β1 intercellular transfer led to increasing Smad2 phosphorylation and improving cell survival by suppressing apoptosis and enhancing cell mobility. Furthermore, sEV‐mediated drug‐resistant transmission by delivering TGF‐β1 was validated using a zebrafish xenograft tumor model. These results elaborated that sEV‐mediated TGF‐β1 intercellular transfer contributes to adriamycin resistance in BCa.     

Concomitant overexpression of mir‐182‐5p and mir‐182‐3p raises the possibility of IL‐17–producing Treg formation in breast cancer by targeting CD3d,ITK, FOXO1, and NFATs: A meta‐analysis and experimental study

T cells are polarized toward regulatory T cells (Tregs) in tumor microenvironment by the shuttling of microRNAs that target T cell–activating signaling pathways. We evaluated the expression of the miR‐182 cluster (miR‐96, 182, and 183) in peripheral blood mononuclear cells (PBMCs) of patients with breast cancer (BC), and T cell polarization by the expression of FOXO1, NFATs, ITK, TCR/CD3 complex, and IL‐2/IL‐2RA. Twenty‐six microRNAs overexpressed in tumor tissues and sera of these patients were extracted by a meta‐analysis. Then, the expression of the miR‐182 cluster was investigated in PBMCs and sera of these patients and correlated with their targets in PBMCs. Finally, miR‐182 was cloned into Jurkat cells to evaluate its effects on T cell polarization. FOXO1, CD3d, ITK, NFATc3, NFATc4, and IL‐2RA were targeted by miR‐182, due to which their expression decreased in PBMCs of patients. Although IL‐6, IL‐17, and TGF‐β increased after miR‐182 transduction, IL‐2 dramatically decreased. We revealed CD4⁺FOXP3⁺ T cell differentiation in the miR‐182–transduced group. Although miR‐182 has inhibitory effects on T cells by the inhibition of FOXO1, TCR/CD3 complex, NFATs, and IL‐2/IL‐2RA signaling pathways, it increases FOXP3, TGF‐β, and IL‐17 expression to possibly drive T cell deviation toward the transitional state of IL‐17–producing Tregs and Treg formation in the end.     

Tumor‐associated macrophage‐derived transforming growth factor‐β promotes colorectal cancer progression through HIF1‐TRIB3 signaling

Tumor‐associated macrophages (TAMs), one of the most common cell components in the tumor microenvironment, have been reported as key contributors to cancer‐related inflammation and enhanced metastatic progression of tumors. To explore the underlying mechanism of TAM‐induced tumor progression, TAMs were isolated from colorectal cancer patients, and the functional interaction with colorectal cancer cells was analyzed. Our study found that coculture of TAMs contributed to a glycolytic state in colorectal cancer, which promoted the stem‐like phenotypes and invasion of tumor cells. TAMs produced the cytokine transforming growth factor‐β to support hypoxia‐inducible factor 1α (HIF1α) expression, thereby upregulating Tribbles pseudokinase 3 (TRIB3) in tumor cells. Elevated expression of TRIB3 resulted in activation of the β‐catenin/Wnt signaling pathway, which eventually enhanced the stem‐like phenotypes and cell invasion in colorectal cancer. Our findings provided evidence that TAMs promoted colorectal cancer progression in a HIF1α/TRIB3‐dependent manner, and blockade of HIF1α signals efficiently improved the outcome of chemotherapy, describing an innovative approach for colorectal cancer treatment.     

Dvl2 facilitates the coordination of NF‐κB and Wnt signaling to promote colitis‐associated colorectal progression

Colitis‐associated colorectal cancer (CAC) arises due to prolonged inflammation and has distinct molecular events compared with sporadic colorectal cancer (CRC). Although inflammatory NF‐κB signaling was activated by pro‐inflammatory cytokines (such as TNFα) in early stages of CAC, Wnt/β‐catenin signaling later appears to function as a key regulator of CAC progression. However, the exact mechanism responsible for the cross‐regulation between these 2 pathways remains unclear. Here, we found reciprocal inhibition between NF‐κB and Wnt/β‐catenin signaling in CAC samples, and the Dvl2, an adaptor protein of Wnt/β‐catenin signaling, is responsible for NF‐κB inhibition. Mechanistically, Dvl2 interacts with the C‐terminus of tumor necrosis factor receptor 1 (TNFRI) and mediates TNFRI endocytosis, leading to NF‐κB signal inhibition. In addition, increased infiltration of the pro‐inflammatory cytokine interleukin‐13 (IL‐13) is responsible for upregulating Dvl2 expression through STAT6. Targeting STAT6 effectively decreases Dvl2 levels and restrains colony formation of cancer cells. These findings demonstrate a unique role for Dvl2 in TNFRI endocytosis, which facilitates the coordination of NF‐κB and Wnt to promote CAC progression.     

Cancer‐associated fibroblast migration in non‐small cell lung cancers is modulated by increased integrin α11 expression

Cancer‐associated fibroblasts (CAFs) regulate cancer progression through the modulation of extracellular matrix (ECM) and cancer cell adhesion. While undergoing a series of phenotypic changes, CAFs control cancer–stroma interactions through integrin receptor signaling. Here, we isolated CAFs from patients with non‐small‐cell lung cancer (NSCLC) and examined their gene expression profiles. We identified collagen type XI α1 (COL11A1), integrin α11 (ITGA11), and the ITGA11 major ligand collagen type I α1 (COL1A1) among the 390 genes that were significantly enriched in NSCLC‐associated CAFs. Increased ITGA11 expression in cancer stroma was correlated with a poor clinical outcome in patients with NSCLC. Increased expression of fibronectin and collagen type I induced ITGA11 expression in CAFs. The cellular migration of CAFs toward collagen type I and fibronectin was promoted via ERK1/2 signaling, independently of the fibronectin receptor integrin α5β1. Additionally, ERK1/2 signaling induced ITGA11 and COL11A1 expression in cancer stroma. We, therefore, propose that targeting ITGA11 and COL11A1 expressing CAFs to block cancer–stroma interactions may serve as a novel, promising anti‐tumor strategy.     

Doxorubicin‐induced senescence promotes stemness and tumorigenicity in EpCAM−/CD133− nonstem cell population in hepatocellular carcinoma cell line,HuH‐7

The therapeutic induction of senescence is a potential means to treat cancer, primarily acting through the induction of a persistent growth‐arrested state in tumors. However, recent studies have indicated that therapy‐induced senescence (TIS) in tumor cells allows for the prolonged survival of a subgroup of cells in a dormant state, with the potential to re‐enter the cell cycle along with an increased stemness gene expression. Residual cells after TIS with increased cancer stem cell phenotype may have profound implications for tumor aggressiveness and disease recurrence. Herein, we investigated senescence‐associated stemness in EpCAM+/CD133+ liver cancer stem cell and EpCAM−/CD133− nonstem cell populations in HuH7 cell line. We demonstrated that treatment with doxorubicin induces senescence in both cell populations, accompanied by a significant increase in the expression of reprogramming genes SOX2, KLF4, and c‐MYC as well as liver stemness‐related genes EpCAM, CK19, and ANXA3 and the multidrug resistance‐related gene ABCG2. Moreover, doxorubicin treatment significantly increased EpCAM + population in nonstem cells indicating senescence‐associated reprogramming of nonstem cell population. Also, Wnt/β‐catenin target genes were increased in these cells, while inhibition of this signaling pathway decreased stem cell gene expression. Importantly, Dox‐treated EpCAM−/CD133− nonstem cells had increased in vivo tumor‐forming ability. In addition, when SASP‐CM from Dox‐treated cells were applied onto hİPSC‐derived hepatocytes, senescence was induced in hepatocytes along with an increased expression of TGF‐β, KLF4, and AXIN2. Importantly, SASP‐CM was not able to induce senescence in Hep3B‐TR cells, a derivative line rendered resistant to TGF‐β signaling. Furthermore, ELISA experiments revealed that the SASP‐CM of Dox‐treated cells contain inflammatory cytokines IL8 and IP10. In summary, our findings further emphasize the importance of carefully dissecting the beneficial and detrimental aspects of prosenescence therapy in HCC and support the potential use of senolytic drugs in HCC treatment in order to eliminate adverse effects of TIS.     

Far‐infrared radiation alleviates cisplatin‐induced vascular damage and impaired circulation via activation of HIF‐1α

Severe vascular damage and complications are often observed in cancer patients during treatment with chemotherapeutic drugs such as cisplatin. Thus, development of potential options to ameliorate the vascular side effects is urgently needed. In this study, the effects and the underlying mechanisms of far‐infrared radiation (FIR) on cisplatin‐induced vascular injury and endothelial cytotoxicity/dysfunction in mice and human umbilical vein endothelial cells (HUVECs) were investigated. An important finding is that the severe vascular stenosis and poor blood flow seen in cisplatin‐treated mice were greatly mitigated by FIR irradiation (30 minutes/day) for 1‐3 days. Moreover, FIR markedly increased the levels of phosphorylation of PI3K and Akt, and VEGF secretion, as well as the expression and the activity of hypoxia‐inducible factor 1α (HIF‐1α) in cisplatin‐treated HUVECs in a promyelocytic leukemia zinc finger protein (PLZF)‐dependent manner. However, FIR‐stimulated endothelial angiogenesis and VEGF release were significantly diminished by transfection with HIF‐1α siRNA. We also confirmed that HIF‐1α, PI3K, and PLZF contribute to the inhibitory effect of FIR on cisplatin‐induced apoptosis in HUVECs. Notably, FIR did not affect the anticancer activity and the HIF‐1α/VEGF cascade in cisplatin‐treated cancer cells under normoxic or hypoxic condition, indicating that the actions of FIR may specifically target endothelial cells. It is the first study to demonstrate that FIR effectively attenuates cisplatin‐induced vascular damage and impaired angiogenesis through activation of HIF‐1α–dependent processes via regulation of PLZF and PI3K/Akt. Taken together, cotreatment with the noninvasive and easily performed FIR has a therapeutic potential to prevent the pathogenesis of vascular complications in cancer patients during cisplatin treatment.     

UBE2T promotes β‐catenin nuclear translocation in hepatocellular carcinoma through MAPK/ERK‐dependent activation

Ubiquitin‐conjugating enzyme E2T (UBE2T) has been implicated in many types of cancer including hepatocellular carcinoma (HCC). Epithelial–mesenchymal transition (EMT) process plays a fundamental role during tumor metastasis and progression. However, the molecular mechanisms underlying EMT in HCC in accordance with UBE2T still remain unknown. In this study, we showed that UBE2T overexpression augmented the oncogenic properties and specifically EMT in HCC cell lines, while its silencing attenuated them. UBE2T affected the activation of EMT‐associated signaling pathways: MAPK/ERK, AKT/mTOR, and Wnt/β‐catenin. In addition, we revealed that the epithelial protein complex of E‐cadherin/β‐catenin, a vital regulator of signal transduction in tumor initiation and progression, was totally disrupted at the cell membrane. In particular, we observed that UBE2T overexpression led to E‐cadherin loss accompanied by a simultaneous elevation of both cytoplasmic and nuclear β‐catenin, while its silencing resulted in a strong E‐cadherin turnover at the cell membrane. Interestingly, chemical inhibition of the MAPK/ERK, AKT/mTOR, and Wnt/β‐catenin signaling pathways demonstrated that the nuclear translocation of β‐catenin and subsequent EMT was enhanced mainly by MAPK/ERK. Collectively, our findings demonstrate the UBE2T/MAPK‐ERK/β‐catenin axis as a critical regulator of cell state transition and EMT in HCC.     

LAMC1 upregulation via TGFβ induces inflammatory cancer‐associated fibroblasts in esophageal squamous cell carcinoma via NF‐κB–CXCL1–STAT3

Cancer‐associated fibroblasts (CAF) are a heterogeneous cell population within the tumor microenvironment,and play an important role in tumor development. By regulating the heterogeneity of CAF, transforming growth factor β (TGFβ) influences tumor development. Here, we explored oncogenes regulated by TGFβ1 that are also involved in signaling pathways and interactions within the tumor microenvironment. We analyzed sequencing data of The Cancer Genome Atlas (TCGA) and our own previously established RNA microarray data ({"type":"entrez-geo","attrs":{"text":"GSE53625","term_id":"53625"}}GSE53625), as well as esophageal squamous cell carcinoma (ESCC) cell lines with or without TGFβ1 stimulation. We then focused on laminin subunit gamma 1 (LAMC1), which was overexpressed in ESCC cells, affecting patient prognosis, which could be upregulated by TGFβ1 through the synergistic activation of SMAD family member 4 (SMAD4) and SP1. LAMC1 directly promoted the proliferation and migration of tumor cells, mainly via Akt–NFκB–MMP9/14 signaling. Additionally, LAMC1 promoted CXCL1 secretion, which stimulated the formation of inflammatory CAF (iCAF) through CXCR2–pSTAT3. Inflammatory CAF promoted tumor progression. In summary, we identified the dual mechanism by which the upregulation of LAMC1 by TGFβ in tumor cells not only promotes ESCC proliferation and migration, but also indirectly induces carcinogenesis by stimulating CXCL1 secretion to promote the formation of iCAF. This finding suggests that LAMC1 could be a potential therapeutic target and prognostic marker for ESCC.     

Cancer‐associated fibroblasts educate normal fibroblasts to facilitate cancer cell spreading and T‐cell suppression

In some tumors, a small number of cancer cells are scattered in a large fibrotic stroma. Here, we demonstrate a novel mechanism for expansion of pro‐tumor fibroblasts via cancer‐associated fibroblast (CAF)‐mediated education of normal fibroblasts (NFs). When NFs were incubated with conditioned medium from CAFs, the resulting CAF‐educated fibroblasts (CEFs) generated reactive oxygen species, which induced NF‐κB‐mediated expression of inflammatory cytokines and the extracellular matrix protein asporin (ASPN), while expression of a common CAF marker gene, α‐SMA, was not increased. ASPN further increased CEF expression of downstream molecules, including indoleamine 2,3‐dioxygenase 1 (IDO‐1), kynureninase (KYNU), and pregnancy‐associated plasma protein‐A (PAPP‐A). These CEFs induce cytocidal effects against CD8⁺ T cells and IGF‐I activation in cancer cells. CEFs were generated without cancer cells by the direct mixture of NFs and CAFs in mouse xenografts, and once CEFs were generated, they sequentially educated NFs, leading to continuous generation of CEFs. In diffuse‐type gastric cancers, ASPN^high/IDO‐1^high/KYNU^high/α‐SMA⁻ CEFs were located at the distal invading front. These CEFs expanded in the fibrotic stroma and caused dissemination of cancer cells. ASPN may therefore be a key molecule in facilitating tumor spreading and T‐cell suppression.