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.     

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.     

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.     

High‐throughput and multi‐phases identification of autoantibodies in diagnosing early‐stage breast cancer and subtypes

Autoantibodies (AAbs) targeted tumor‐associated antigens (TAAs) have the potential for early detection of breast cancer. Here, 574 early‐stage breast cancer (ES‐BC) patients containing 4 subtypes (Luminal A, Luminal B, HER2+, TN), 126 benign breast disease (BBD) patients, and 199 normal healthy controls (NHC) were separated into three‐phases to discover, verify, and validate AAbs. In discovery phase using high‐throughput protein microarray, 37 AAbs with sensitivity of 31.25%‐86.25% and specificity over 73% in ES‐BC, and 40 AAbs with different positive rates between subtypes were identified as candidates. In verification phase, 18 AAbs were significantly increased compared with the Control (BBD and NHC) in focused array. Ten out of 18 AAbs exhibited a significant difference between subtypes (P < .05). In ELISA validation phase, 5 novel AAbs (anti‐{"type":"entrez-nucleotide","attrs":{"text":"KJ901215","term_id":"649134855","term_text":"KJ901215"}}KJ901215, ‐FAM49B, ‐HYI, ‐GARS, ‐CRLF3) exhibited significantly higher levels in ES‐BC compared with BBD/NHC (P < .05). The sensitivities of individual AAb and a 5‐AAbs panel were 20.41%‐28.57% and 38.78%, whereas the specificities were over 90% and 85.94%. Simultaneously, 4 AAbs except anti‐GARS differed significantly between TN and non‐TN subtype (P < .05). We constructed 3 random forest classifier models based on AAbs to discriminant ES‐BC from Control or BBD, and to discern TN subtype, which yielded an area under the curve of 0.870, 0.860, and 0.875, respectively. Biological interaction analysis revealed 4 TAAs, except for {"type":"entrez-nucleotide","attrs":{"text":"KJ901215","term_id":"649134855","term_text":"KJ901215"}}KJ901215, that were associated with well known proteins of BC. This study discovered and stepwise validated 5 novel AAbs with the potential to diagnose ES‐BC and discern TN subtype, indicating easy‐to‐detect and minimally invasive diagnostic value of serum AAbs ahead of biopsy for future application.     

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.     

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.     

Anti‐pyroptotic function of TGF‐β is suppressed by a synthetic dsRNA analogue in triple negative breast cancer cells

Development of innovative therapeutic modalities would address an unmet clinical need in the treatment of triple negative breast cancer (TNBC). Activation of retinoic acid‐inducible gene‐I (RIG‐I)‐like receptors (RLRs) such as melanoma differentiation‐associated gene 5 (MDA5) and RIG‐I in cancer cells is suggested to suppress tumor progression by inducing cell death. Transfection of polyI:C, a conventionally used synthetic double‐stranded RNA (dsRNA) analogue that activates RLRs, has been evaluated in clinical trials. However, detailed mechanisms of tumor suppression by RLRs, especially interactions with other signaling pathways, remain elusive. Here, we showed that transfection of polyI:C suppressed transforming growth factor‐β (TGF‐β) signaling in a MDA5‐ and RIG‐I‐dependent manner. We found that suppression of TGF‐β signaling by polyI:C promoted cancer cell death, which was attenuated by forced expression of constitutively active Smad3. More detailed analysis suggested that cell death by polyI:C transfection exhibited characteristics of pyroptosis, which is distinct from apoptosis. Therapeutic efficacy of polyI:C transfection was also demonstrated using a mouse model. These results indicated that intratumor administration of polyI:C and related dsRNA analogues may be promising treatments for TNBC through inhibition of the anti‐pyroptotic function of TGF‐β.     

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.     

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.     

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.     

27‐Hydroxycholesterol promotes metastasis by SULT2A1‐dependent alteration in hepatocellular carcinoma

Oxysterol metabolism plays an important role in the initiation and development of various tumors. However, little is known that the metabolic alternation can promote the metastasis of hepatocellular carcinoma (HCC). In this study, we identify the sulfotransferase family 2A member 1 (SULT2A1) to 27‐hydroxycholesterol (27‐OHC) metabolic axis as playing a critical role in HCC metastasis. The level of 27‐OHC closely corresponded with HCC metastasis instead of proliferation in vitro and in vivo. Also, the expression of SULT2A1 is extremely downregulated in human HCC tissues and is correlated with poor prognosis and tumor metastasis. Gain‐ and loss‐of‐function studies reveal that SULT2A1 suppresses the metastasis of HCC by regulating the level of 27‐OHC. Further mechanistic studies indicated that SULT2A1‐dependent alternation of 27‐OHC activates the nuclear factor‐κB signaling pathway and promotes HCC metastasis by enhancing Twist1 expression and epithelial–mesenchymal transition. In conclusion, our findings indicate the relationship between the metabolism of 27‐OHC and the metastasis of HCC. Moreover, SULT2A1 could act as a potential prognostic biomarker and a therapeutic target for preventing HCC metastasis.     

Targeted inhibition of ERα signaling and PIP5K1α/Akt pathways in castration‐resistant prostate cancer

Selective ERα modulator, tamoxifen, is well tolerated in a heavily pretreated castration‐resistant prostate cancer (PCa) patient cohort. However, its targeted gene network and whether expression of intratumor ERα due to androgen deprivation therapy (ADT) may play a role in PCa progression is unknown. In this study, we examined the inhibitory effect of tamoxifen on castration‐resistant PCa in vitro and in vivo. We found that tamoxifen is a potent compound that induced a high degree of apoptosis and significantly suppressed growth of xenograft tumors in mice, at a degree comparable to ISA‐2011B, an inhibitor of PIP5K1α that acts upstream of PI3K/AKT survival signaling pathway. Moreover, depletion of tumor‐associated macrophages using clodronate in combination with tamoxifen increased inhibitory effect of tamoxifen on aggressive prostate tumors. We showed that both tamoxifen and ISA‐2011B exert their on‐target effects on prostate cancer cells by targeting cyclin D1 and PIP5K1α/AKT network and the interlinked estrogen signaling. Combination treatment using tamoxifen together with ISA‐2011B resulted in tumor regression and had superior inhibitory effect compared with that of tamoxifen or ISA‐2011B alone. We have identified sets of genes that are specifically targeted by tamoxifen, ISA‐2011B or combination of both agents by RNA‐seq. We discovered that alterations in unique gene signatures, in particular estrogen‐related marker genes are associated with poor patient disease‐free survival. We further showed that ERα interacted with PIP5K1α through formation of protein complexes in the nucleus, suggesting a functional link. Our finding is the first to suggest a new therapeutic potential to inhibit or utilize the mechanisms related to ERα, PIP5K1α/AKT network, and MMP9/VEGF signaling axis, providing a strategy to treat castration‐resistant ER‐positive subtype of prostate cancer tumors with metastatic potential.

Abbreviations

CRPC

castration‐resistant prostate cancer

DHT

dihydrotestosterone

E2

estradiol

ERα

estrogen receptor alpha

GO

gene ontology

NG‐CHM

Next‐Generation Clustered Heatmaps

PCa

prostate cancer

TMAs

tissue microarrays