Collective cancer cell invasion in contact with fibroblasts through integrin‐α5β1/fibronectin interaction in collagen matrix

Interaction of cancer cells with cancer‐associated fibroblasts (CAFs) plays critical roles in tumor progression. Recently we proposed a new tumor invasion mechanism in which invasive cancer cells individually migrate on elongate protrusions of CAFs (CAF fibers) in 3‐D collagen matrix. In this mechanism, cancer cells interact with fibronectin fibrils assembled on CAFs mainly through integrin‐α5β1. Here we tested whether this mechanism is applicable to the collective invasion of cancer cells, using two E‐cadherin‐expressing adenocarcinoma cell lines, DLD‐1 (colon) and MCF‐7 (breast). When hybrid spheroids of DLD‐1 cells with CAFs were embedded into collagen gel, DLD‐1 cells collectively but very slowly migrated through the collagen matrix in contact with CAFs. Epidermal growth factor and tumor necrosis factor‐α promoted the collective invasion, possibly by reducing the E‐cadherin junction, as did the transforming growth factor‐β inhibitor SB431542 by stimulating the outgrowth of CAFs. Transforming growth factor‐β itself inhibited the cancer cell invasion. Efficient collective invasion of DLD‐1 cells required large CAF fibers or their assembly as stable adhesion substrates. Experiments with function‐blocking Abs and siRNAs confirmed that DLD‐1 cells adhered to fibronectin fibrils on CAFs mainly through integrin‐α5β1. Anti‐E‐cadherin Ab promoted the single cell invasion of DLD‐1 cells by dissociating the E‐cadherin junction. Although the binding affinity of MCF‐7 cells to CAFs was lower than DLD‐1, they also collectively invaded the collagen matrix in a similar fashion to DLD‐1 cells. Our results suggest that the direct interaction with CAFs, as well as environmental cytokines, contributes to the collective invasion of cancers.     

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.     

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.     

PELI1 promotes radiotherapy sensitivity by inhibiting noncanonical NF‐κB in esophageal squamous cancer

The low sensitivity of radiotherapy is the main cause of tumor tolerance against ionizing radiation (IR). However, the molecular mechanisms by which radiosensitivity is controlled remain elusive. Here, we observed that high expression of pellino E3 ubiquitin protein ligase 1 (PELI1) was correlated with improved prognosis in human esophageal squamous cell carcinoma stage III patients that received adjuvant radiotherapy. Moreover, we found PELI1‐mediated IR‐induced tumor cell apoptosis in vivo and in vitro. Mechanistically, PELI1 mediated the lysine 48 (Lys48)–linked polyubiquitination and degradation of NF‐κB–inducing kinase (NIK; also known as MAP3K14), the master kinase of the noncanonical NF‐κB pathway, thereby inhibiting IR‐induced activation of the noncanonical NF‐κB signaling pathway during radiotherapy. As a consequence, PELI1 inhibited the noncanonical NF‐κB–induced expression of the anti‐apoptotic gene BCL2 like 1 (Bclxl; also known as BCL2L1), leading to an enhancement of the IR‐induced apoptosis signaling pathway and ultimately promoting IR‐induced apoptosis in tumor cells. Therefore, Bclxl or NIK knockdown abolished the apoptosis‐resistant effect in PELI1‐knockdown tumor cells after radiotherapy. These findings establish PELI1 as a critical tumor intrinsic regulator in controlling the sensitivity of tumor cells to radiotherapy through modulating IR‐induced noncanonical NF‐κB expression.     

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.     

Long noncoding RNA LINC01578 drives colon cancer metastasis through a positive feedback loop with the NF‐κB/YY1 axis

Metastasis accounts for poor prognosis of cancers and related deaths. Accumulating evidence has shown that long noncoding RNAs (lncRNAs) play critical roles in several types of cancer. However, which lncRNAs contribute to metastasis of colon cancer is still largely unknown. In this study, we found that lncRNA LINC01578 was correlated with metastasis and poor prognosis of colon cancer. LINC01578 was upregulated in colon cancer, associated with metastasis, advanced clinical stages, poor overall survival, disease‐specific survival, and disease‐free survival. Gain‐of‐function and loss‐of‐function assays revealed that LINC01578 enhanced colon cancer cell viability and mobility in vitro and colon cancer liver metastasis in vivo. Mechanistically, nuclear factor kappa B (NF‐κB) and Yin Yang 1 (YY1) directly bound to the LINC01578 promoter, enhanced its activity, and activated LINC01578 expression. LINC01578 was shown to be a chromatin‐bound lncRNA, which directly bound NFKBIB promoter. Furthermore, LINC01578 interacted with and recruited EZH2 to NFKBIB promoter and further repressed NFKBIB expression, thereby activating NF‐κB signaling. Through activation of NF‐κB, LINC01578 further upregulated YY1 expression. Through activation of the NF‐κB/YY1 axis, LINC01578 in turn enhanced its own promoter activity, suggesting that LINC01578 and NF‐κB/YY1 formed a positive feedback loop. Blocking NF‐κB signaling abolished the oncogenic roles of LINC01578 in colon cancer. Furthermore, the expression levels of LINC01578, NFKBIB, and YY1 were correlated in clinical tissues. Collectively, this study demonstrated that LINC01578 promoted colon cancer metastasis via forming a positive feedback loop with NF‐κB/YY1 and suggested that LINC01578 represents a potential prognostic biomarker and therapeutic target for colon cancer metastasis.

Abbreviations

ChIP

chromatin immunoprecipitation

ChIRP

chromatin isolation by RNA purification

COAD

colon adenocarcinoma

CPAT

Coding‐Potential Assessment Tool

CPC

coding potential calculator

DFS

disease‐free survival

DSS

disease‐specific survival

EdU

5‐ethynyl‐2''‐deoxyuridine

H&E

hematoxylin and eosin

HR

hazard ratio

IHC

immunohistochemistry

IKK

IκB kinase

IκB

inhibitory κB

lncRNAs

long noncoding RNAs

NC

negative control

NCBI

National Center for Biotechnology Information

NF‐κB

nuclear factor kappa B

qRT‐PCR

quantitative real‐time polymerase chain reaction

RIP

RNA immunoprecipitation

RPISeq

RNA‐Protein Interaction Prediction

TCGA

The Cancer Genome Atlas

TNF

tumor necrosis factor

TUNEL

TdT‐mediated dUTP Nick‐End Labeling

YY1

Yin Yang 1

     

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.     

Periostin+cancer‐associated fibroblasts promote lymph node metastasis by impairing the lymphatic endothelial barriers in cervical squamous cell carcinoma

Lymph node metastasis (LNM), a critical prognostic determinant in cancer patients, is critically influenced by the presence of numerous heterogeneous cancer‐associated fibroblasts (CAFs) in the tumor microenvironment. However, the phenotypes and characteristics of the various pro‐metastatic CAF subsets in cervical squamous cell carcinoma (CSCC) remain unknown. Here, we describe a CAF subpopulation with elevated periostin expression (periostin⁺CAFs), located in the primary tumor sites and metastatic lymph nodes, that positively correlated with LNM and poor survival in CSCC patients. Mechanistically, periostin⁺CAFs impaired lymphatic endothelial barriers by activating the integrin‐FAK/Src‐VE‐cadherin signaling pathway in lymphatic endothelial cells and consequently enhanced metastatic dissemination. In contrast, inhibition of the FAK/Src signaling pathway alleviated periostin‐induced lymphatic endothelial barrier dysfunction and its related effects. Notably, periostin^‐CAFs were incapable of impairing endothelial barrier integrity, which may explain the occurrence of CAF‐enriched cases without LNM. In conclusion, we identified a specific periostin⁺CAF subset that promotes LNM in CSCC, mainly by impairing the lymphatic endothelial barriers, thus providing the basis for potential stromal fibroblast‐targeted interventions that block CAF‐dependent metastasis.

Abbreviations

CAFs

cancer‐associated fibroblasts

CM

conditioned medium

CSCC

cervical squamous cell carcinoma

HDLECs

human dermal lymphatic endothelial cells

LN

lymph node

LNM

lymph node metastasis

LVs

lymphatic vessels

MFI

mean fluorescence intensity

NC

negative control

NOFs

normal fibroblasts

SFM

serum‐free media

TAMs

tumor‐associated macrophages

TEM

transmission electron microscopy

TME

tumor microenvironment

     

Activated macrophages promote invasion by early colorectal cancer via an interleukin 1β‐serum amyloid A1 axis

Submucosal invasion and lymph node metastasis are important issues affecting treatment options for early colorectal cancer (CRC). In this study, we aimed to unravel the molecular mechanism underlying the invasiveness of early CRCs. We performed RNA‐sequencing (RNA‐seq) with poorly differentiated components (PORs) and their normal counterparts isolated from T1 CRC tissues and detected significant upregulation of serum amyloid A1 (SAA1) in PORs. Immunohistochemical analysis revealed that SAA1 was specifically expressed in PORs at the invasive front of T1b CRCs. Upregulation of SAA1 in CRC cells promoted cell migration and invasion. Coculture experiments using CRC cell lines and THP‐1 cells suggested that interleukin 1β (IL‐1β) produced by macrophages induces SAA1 expression in CRC cells. Induction of SAA1 and promotion of CRC cell migration and invasion by macrophages were inhibited by blocking IL‐1β. These findings were supported by immunohistochemical analysis of primary T1 CRCs showing accumulation of M1‐like/M2‐like macrophages at SAA1‐positive invasive front regions. Moreover, SAA1 produced by CRC cells stimulated upregulation of matrix metalloproteinase‐9 in macrophages. Our data suggest that tumor‐associated macrophages at the invasive front of early CRCs promote cancer cell migration and invasion through induction of SAA1 and that SAA1 may be a predictive biomarker and a useful therapeutic target.     

Adoptive transfer of immature myeloid cells lacking NF‐κB p50 (p50‐IMC) impedes the growth of MHC‐matched high‐risk neuroblastoma

High‐risk neuroblastomas harbor abundant myeloid cells that suppress antitumor immunity and support tumor growth. Macrophages lacking the inhibitory NF‐κB p50 subunit adopt a pro‐inflammatory phenotype. We now report that murine 9464D neuroblastoma cells, which express high levels of exogenous MYCN, grow slower in syngeneic p50(f/f);Lys‐Cre mice that lack p50 in macrophages and neutrophils, compared with p50(f/f) littermates. Tumors in p50(f/f);Lys‐Cre mice possess increased numbers of total and activated CD4⁺ and CD8⁺ T cells, and depletion of both of these T‐cell populations accelerates tumor growth. Anti‐PD‐1 T‐cell checkpoint blockade, or DNA methyltransferase and histone deacetylase inhibition, further slows tumor growth. In addition, adoptive transfer of immature myeloid cells lacking NF‐κB p50 (p50‐IMC), generated either from the bone marrow of p50^−/− mice or via nucleofection of a p50 sgRNA:Cas9 complex into wild‐type hematopoietic progenitors, also slowed growth of MHC‐matched 9464D tumors but not of MHC‐mismatched Neuro2A tumors. These findings further validate the utility of targeting myeloid NF‐κB p50 as a strategy for cancer therapy and demonstrate activity of p50‐IMC generated by gene editing of syngeneic marrow cells, a cell product relevant to clinical translation.     

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.     

The CD226‐ERK1/2‐LAMP1 pathway is an important mechanism for Vγ9Vδ2 T cell cytotoxicity against chemotherapy‐resistant acute myeloid leukemia blasts and leukemia stem cells

Vγ9Vδ2 T cells are attractive effector cells for immunotherapy with potent cytotoxic activity against a variety of malignant cells. However, the effect of Vγ9Vδ2 T cells on chemotherapy‐resistant acute myeloid leukemia (AML) blasts, especially highly refractory leukemia stem cells (LSCs) is still unknown. In this study, we investigated the effect of cytotoxicity of allogeneic Vγ9Vδ2 T cells on chemotherapy‐resistant AML cell lines, as well as on primary AML blasts and LSCs obtained from refractory AML patients. The results indicated that Vγ9Vδ2 T cells can efficiently kill drug‐resistant AML cell lines in vitro and in vivo, and the sensitivity of AML cells to Vγ9Vδ2 T cell–mediated cytotoxicity is not influenced by the sensitivity of AML cells to chemotherapy. We further found that Vγ9Vδ2 T cells exhibited a comparable effect of cytotoxicity against LSCs to primary AML blasts. More importantly, we revealed that the CD226–extracellular signal–regulatory kinase1/2 (ERK1/2)–lysosome‐associated membrane protein 1 (LAMP1) pathway is an important mechanism for Vγ9Vδ2 T cell–induced cytotoxicity against AML cells. First, Vγ9Vδ2 T cells recognized AML cells by receptor‐ligand interaction of CD226–Nectin‐2, which then induced ERK1/2 phosphorylation in Vγ9Vδ2 T cells. Finally, triggering the movement of lytic granules toward AML cells induced cytolysis of AML cells. The expression level of Nectin‐2 may be used as a novel marker to predict the susceptibility/resistance of AML cells to Vγ9Vδ2 T cell treatment.     

Long noncoding RNA DANCR confers cytarabine resistance in acute myeloid leukemia by activating autophagy via the miR‐874‐3P/ATG16L1 axis

Autophagy is an important mechanism involved in the regulation of acute myeloid leukemia (AML) chemoresistance. The long noncoding RNA (lncRNA) differentiation antagonizing non‐protein coding RNA (DANCR) exhibits oncogenic activity in several types of human cancers, including AML, but it remains unclear whether it regulates autophagy and chemoresistance in AML. We report here that cytarabine (Ara‐C) treatment elevates DANCR expression in human AML cells. In addition, DANCR overexpression confers and its knockdown diminishes Ara‐C resistance in human AML cells, suggesting that DANCR positively regulates AML chemoresistance to Ara‐C. Moreover, DANCR promotes autophagy in Ara‐C‐treated human AML cells and acts as a sponge to decrease miR‐20a‐5p expression, thereby upregulating the expression of ATG16L1, a critical component of the autophagy machinery. Importantly, ATG16L1 silencing abrogates DANCR‐promoted autophagy and markedly restores DANCR‐conferred Ara‐C resistance, suggesting that DANCR promotes MIR‐874‐3P/ATG16L1 axis‐regulated autophagy to confer Ara‐C resistance in human AML cells. Together, this study identifies DANCR as a positive regulator of Ara‐C resistance in human AML cells, suggesting this lncRNA as a potential target for overcoming Ara‐C resistance in AML chemotherapy.