Rab37 mediates trafficking and membrane presentation of PD-1 to sustain T cell exhaustion in lung cancer.

BACKGROUND: Programmed cell death protein 1 (PD-1) is an immune checkpoint receptor expressed on the surface of T cells. High expression of PD-1 leads to T-cell dysfunction in the tumor microenvironment (TME). However, the mechanism of intracellular trafficking and plasma membrane presentation of PD-1 remains unclear. METHODS: Multiple databases of lung cancer patients were integratively analyzed to screen Rab proteins and potential immune-related signaling pathways. Imaging and various biochemical assays were performed in Jurkat T cells, splenocytes, and human peripheral blood mononuclear cells (PBMCs). Rab37 knockout mice and specimens of lung cancer patients were used to validate the concept. RESULTS: Here, we identify novel mechanisms of intracellular trafficking and plasma membrane presentation of PD-1 mediated by Rab37 small GTPase to sustain T cell exhaustion, thereby leading to poor patient outcome. PD-1 colocalized with Rab37-specific vesicles of T cells in a GTP-dependent manner whereby Rab37 mediated dynamic trafficking and membrane presentation of PD-1. However, glycosylation mutant PD-1 delayed cargo recruitment to the Rab37 vesicles, thus stalling membrane presentation. Notably, T cell proliferation and activity were upregulated in tumor-infiltrating T cells from the tumor-bearing Rab37 knockout mice compared to those from wild type. Clinically, the multiplex immunofluorescence-immunohistochemical assay indicated that patients with high Rab37+/PD-1+/TIM3+/CD8+ tumor infiltrating T cell profile correlated with advanced tumor stages and poor overall survival. Moreover, human PBMCs from patients demonstrated high expression of Rab37, which positively correlated with elevated levels of PD-1+ and TIM3+ in CD8+ T cells exhibiting reduced tumoricidal activity. CONCLUSIONS: Our results provide the first evidence that Rab37 small GTPase mediates trafficking and membrane presentation of PD-1 to sustain T cell exhaustion, and the tumor promoting function of Rab37/PD-1 axis in T cells of TME in lung cancer. The expression profile of Rab37high/PD-1high/TIM3high in tumor-infiltrating CD8+ T cells is a biomarker for poor prognosis in lung cancer patients.

PTPN23 ubiquitination by WDR4 suppresses EGFR and c-MET degradation to define a lung cancer therapeutic target.

Aberrant overexpression or activation of EGFR drives the development of non-small cell lung cancer (NSCLC) and acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) by secondary EGFR mutations or c-MET amplification/activation remains as a major hurdle for NSCLC treatment. We previously identified WDR4 as a substrate adaptor of Cullin 4 ubiquitin ligase and an association of WDR4 high expression with poor prognosis of lung cancer. Here, using an unbiased ubiquitylome analysis, we uncover PTPN23, a component of the ESCRT complex, as a substrate of WDR4-based ubiquitin ligase. WDR4-mediated PTPN23 ubiquitination leads to its proteasomal degradation, thereby suppressing lysosome trafficking and degradation of wild type EGFR, EGFR mutant, and c-MET. Through this mechanism, WDR4 sustains EGFR and c-MET signaling to promote NSCLC proliferation, migration, invasion, stemness, and metastasis. Clinically, PTPN23 is downregulated in lung cancer and its low expression correlates with WDR4 high expression and poor prognosis. Targeting WDR4-mediated PTPN23 ubiquitination by a peptide that competes with PTPN23 for binding WDR4 promotes EGFR and c-MET degradation to block the growth and progression of EGFR TKI-resistant NSCLC. These findings identify a central role of WDR4/PTPN23 axis in EGFR and c-MET trafficking and a potential therapeutic target for treating EGFR TKI-resistant NSCLC.

Nutlin-3 acts as a DNA methyltransferase inhibitor to sensitize esophageal cancer to chemoradiation.

Esophageal squamous cell carcinoma (ESCC) is highly resistant to chemoradiation therapy. We aimed to examine whether Nutlin-3, a molecule that suppresses murine double min 2 (MDM2)-mediated p53 and Retinoblastoma (RB) protein degradation leading to downregulation of DNA methyltransferases (DNMTs), can be a novel therapeutic agent for ESCC. We used wild-type and chemoradiation-resistant ESCC cell lines in this study. The expression of DNMTs, p53 and RB, and methylation level of tumor suppressor genes (TSG) were analyzed upon Nutlin-3 treatment. The antitumor efficacy of Nutlin-3 was investigated in ESCC cell lines and xenograft tumor model. TSG protein expression was checked in the excised tumor tissue. Nutlin-3 induced upregulation of p53 and RB and downregulation of DNMTs proteins in the chemoradiation-resistant and aggressive ESCC cells. The methylation level of TSGs was decreased by Nutlin-3. Nutlin-3 inhibits clonogenic growth of ESCC cells and exerts a synergistic cytotoxic-effect when combined with chemotherapeutic agent cisplatin. Moreover, xenograft tumor growth in SCID mice was suppressed by Nutlin-3. The protein expression level of DNMTs was downregulated, and that of TSGs was upregulated by Nutlin-3 treatment in the excised tumor tissue. In conclusion, Nutlin-3 is a potential therapeutic agent that can potentiate the treatment efficacy of chemoradiation-resistant ESCC.

Dysregulation of SOX17/NRF2 axis confers chemoradiotherapy resistance and emerges as a novel therapeutic target in esophageal squamous cell carcinoma.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is the sixth leading cause of cancer-associated death worldwide with a dismal overall 5-year survival rate of less than 20%. The standard first-line therapy for advanced ESCC is concomitant chemo-radiation therapy (CCRT); however, patients usually develop resistance, resulting in unfavorable outcomes. Therefore, it is urgent to identify the mechanisms underlying CCRT resistance and develop effective treatment strategies. METHODS: Patients' endoscopic biopsy tumor tissues obtained before CCRT treatment were used to perform RNA-seq and GSEA analysis. Immunohistochemical (IHC) staining, chromatin immunoprecipitation (ChIP), and promoter reporter analyses were conducted to investigate the relationship between SOX17 and NRF2. Xenograft mouse models were used to study the role of SOX17/NRF2 axis in tumor growth and the efficacy of carboxymethyl cellulose-coated zero-valent-iron (ZVI@CMC). RESULTS: In this study, a notable gene expression signature associated with NRF2 activation was observed in the poor CCRT responders. Further, IHC staining of endoscopic biopsy of 164 ESCC patients revealed an inverse correlation between NRF2 and SOX17, a tumor-suppressive transcription factor with low expression in ESCC due to promoter hypermethylation. Using ChIP and promoter reporter analyses, we demonstrated that SOX17 was a novel upstream transcriptional suppressor of NRF2. In particular, SOX17low/NRF2high nuclear level significantly correlated with poor CCRT response and poor survival, indicating that the dysregulation of SOX17/NRF2 axis played a pivotal role in CCRT resistance and tumor progression. Notably, the in-house developed nanoparticle ZVI@CMC functioned as an inhibitor of DNA methyltransferases to restore expression of SOX17 that downregulated NRF2, thereby overcoming the resistance in ESCC. Additionally, the combination of ZVI@CMC with radiation treatment significantly augmented anticancer efficacy to inhibit tumor growth in CCRT resistant cancer. CONCLUSION: This study identifies a novel SOX17low/NRF2high signature in ESCC patients with poor prognosis, recognizes SOX17 as a transcriptional repressor of NRF2, and provides a promising strategy targeting SOX17/NRF2 axis to overcome resistance.

Recent advances in conventional and unconventional vesicular secretion pathways in the tumor microenvironment.

All cells in the changing tumor microenvironment (TME) need a class of checkpoints to regulate the balance among exocytosis, endocytosis, recycling and degradation. The vesicular trafficking and secretion pathways regulated by the small Rab GTPases and their effectors convey cell growth and migration signals and function as meditators of intercellular communication and molecular transfer. Recent advances suggest that Rab proteins govern conventional and unconventional vesicular secretion pathways by trafficking widely diverse cargoes and substrates in remodeling TME. The mechanisms underlying the regulation of conventional and unconventional vesicular secretion pathways, their action modes and impacts on the cancer and stromal cells have been the focus of much attention for the past two decades. In this review, we discuss the current understanding of vesicular secretion pathways in TME. We begin with an overview of the structure, regulation, substrate recognition and subcellular localization of vesicular secretion pathways. We then systematically discuss how the three fundamental vesicular secretion processes respond to extracellular cues in TME. These processes are the conventional protein secretion via the endoplasmic reticulum-Golgi apparatus route and two types of unconventional protein secretion via extracellular vesicles and secretory autophagy. The latest advances and future directions in vesicular secretion-involved interplays between tumor cells, stromal cell and host immunity are also described.

Targeting protumor factor chitinase-3-like-1 secreted by Rab37 vesicles for cancer immunotherapy.

Background: Chitinase 3-like-1 (CHI3L1) is a secretion glycoprotein associated with the immunosuppressive tumor microenvironment (TME). The secretory mode of CHI3L1 makes it a promising target for cancer treatment. We have previously reported that Rab37 small GTPase mediates secretion of IL-6 in macrophages to promote cancer progression, whereas the roles of Rab37 in the intracellular trafficking and exocytosis of CHI3L1 are unclear. Methods: We examined the concentration of CHI3L1 in the culture medium of splenocytes and bone marrow derived macrophages (BMDMs) from wild-type or Rab37 knockout mice, and macrophage or T cell lines expressing wild type, active GTP-bound or inactive GDP-bound Rab37. Vesicle isolation, total internal reflection fluorescence microscopy, and real-time confocal microscopy were conducted. We developed polyclonal neutralizing-CHI3L1 antibodies (nCHI3L1 Abs) to validate the therapeutic efficacy in orthotopic lung, pancreas and colon cancer allograft models. Multiplex fluorescence immunohistochemistry was performed to detect the protein level of Rab37 and CHI3L1, and localization of the tumor-infiltrating immune cells in allografts from mice or tumor specimens from cancer patients. Results: We demonstrate a novel secretion mode of CHI3L1 mediated by the small GTPase Rab37 in T cells and macrophages. Rab37 mediated CHI3L1 intracellular vesicle trafficking and exocytosis in a GTP-dependent manner, which is abolished in the splenocytes and BMDMs from Rab37 knockout mice and attenuated in macrophage or T cell lines expressing the inactive Rab37. The secreted CHI3L1 activated AKT, ß-catenin and NF-κB signal pathways in cancer cells and macrophages to foster a protumor TME characterized by activating M2 macrophages and increasing the population of regulatory T cells. Our developed nCHI3L1 Abs showed the dual properties of reducing tumor growth/metastases and eliciting an immunostimulatory TME in syngeneic orthotopic lung, pancreas and colon tumor models. Clinically, high plasma level or intratumoral expression of CHI3L1 correlated with poor survival in 161 lung cancer, 155 pancreatic cancer and 180 colon cancer patients. Conclusions: These results provide the first evidence that Rab37 mediates CHI3L1 secretion in immune cells and highlight nCHI3L1 Abs that can simultaneously target both cancer cells and tumor microenvironment.

Carboxypeptidase E mRNA: Overexpression predicts recurrence and death in lung adenocarcinoma cancer patients.

BACKGROUND: Effective biomarkers for prediction of recurrence of lung adenocarcinoma cancer (LADC) patients are needed to determine treatment strategies post-surgery to improve outcome. OBJECTIVE: This study evaluates the efficacy of carboxypeptidase E (CPE) mRNA including its splice isoforms, CPE-ΔN, as a biomarker for predicting recurrence in adenocarcinoma patients. METHODS: RNA was extracted from resected tumors from 86 patients with different stages of non-small cell LADC. cDNA was synthesized and qRT-PCR carried out to determine the copy numbers of CPE/CPE-ΔN mRNA. Patients were followed for 7 years post-tumor resection to determine recurrence and death. RESULTS: ROC curve analysis showed the overall AUC for CPE/CPE-ΔN copy number was 0.563 in predicting recurrence and 0.562 in predicting death. Kaplan-Meier survival analysis showed statistical difference (p= 0.018), indicating that patients with high CPE/CPE-ΔN copy numbers had a shorter time of disease-free survival and also shorter time to death (p= 0.035). Subgroup analyses showed that association of disease-free survival time with CPE/CPE-ΔN copy number was stronger among stage I and II LADC patients (p= 0.047). CONCLUSIONS: CPE/CPE-ΔN mRNA is a potentially useful biomarker for predicting recurrence and death in LADC patients, especially in identifying patients at high risk of recurrence at early stages I and II.

Converged Rab37/IL-6 trafficking and STAT3/PD-1 transcription axes elicit an immunosuppressive lung tumor microenvironment.

Background: Increased IL-6 level, M2 macrophages and PD-1+CD8+ T cells in tumor microenvironments (TME) have been identified to correlate with resistance to checkpoint blockade immunotherapy, yet the mechanism remains poorly understood. Rab small GTPase-mediated trafficking of cytokines is critical in immuno-modulation. We have previously reported dysregulation of Rab37 in lung cancer cells, whereas the roles of Rab37 in tumor-infiltrating immune cells and cancer immunotherapy are unclear. Methods: The tumor growth of the syngeneic mouse allograft in wild type or Rab37 knockout mice was analyzed. Imaging analyses and vesicle isolation were conducted to determine Rab37-mediated IL-6 secretion. STAT3 binding sites at PD-1 promoter in T cells were identified by chromatin immunoprecipitation assay. Multiplex fluorescence immunohistochemistry was performed to detect the protein level of Rab37, IL-6 and PD-1 and localization of the tumor-infiltrating immune cells in allografts from mice or tumor specimens from lung cancer patients. Results: We revealed that Rab37 regulates the secretion of IL-6 in a GTPase-dependent manner in macrophages to trigger M2 polarization. Macrophage-derived IL-6 promotes STAT3-dependent PD-1 mRNA expression in CD8+ T cells. Clinically, tumors with high stromal Rab37 and IL-6 expression coincide with tumor infiltrating M2-macrophages and PD1+CD8+ T cells that predicts poor prognosis in lung cancer patients. In addition, lung cancer patients with an increase in plasma IL-6 level are found to be associated with immunotherapeutic resistance. Importantly, combined blockade of IL-6 and CTLA-4 improves survival of tumor-bearing mice by reducing infiltration of PD1+CD8+ T cells and M2 macrophages in TME. Conclusions: Rab37/IL-6 trafficking pathway links with IL-6/STAT3/PD-1 transcription regulation to foster an immunosuppressive TME and combined IL-6/CTLA-4 blockade therapy exerts potent anti-tumor efficacy.

Associations among phthalate exposure, DNA methylation of TSLP, and childhood allergy.

BACKGROUND: Dysregulation of thymic stromal lymphopoietin (TSLP) expressions is linked to asthma and allergic disease. Exposure to phthalate esters, a widely used plasticizer, is associated with respiratory and allergic morbidity. Dibutyl phthalate (DBP) causes TSLP upregulation in the skin. In addition, phthalate exposure is associated with changes in environmentally induced DNA methylation, which might cause phenotypic heterogeneity. This study examined the DNA methylation of the TSLP gene to determine the potential mechanism between phthalate exposure and allergic diseases. RESULTS: Among all evaluated, only benzyl butyl phthalate (BBzP) in the settled dusts were negatively correlated with the methylation levels of TSLP and positively associated with children's respiratory symptoms. The results revealed that every unit increase in BBzP concentration in the settled dust was associated with a 1.75% decrease in the methylation level on upstream 775 bp from the transcription start site (TSS) of TSLP (ß = - 1.75, p = 0.015) after adjustment for child's sex, age, BMI, parents' smoking status, allergic history, and education levels, PM2.5, formaldehyde, temperature; and relative humidity. Moreover, every percentage increase in the methylation level was associated with a 20% decrease in the risk of morning respiratory symptoms in the children (OR 0.80, 95% CI 0.65-0.99). CONCLUSIONS: Exposure to BBzP in settled dust might increase children's respiratory symptoms in the morning through decreasing TSLP methylation. Therefore, the exposure to BBzP should be reduced especially for the children already having allergic diseases.

Dysregulated Kras/YY1/ZNF322A/Shh transcriptional axis enhances neo-angiogenesis to promote lung cancer progression.

Angiogenesis enhances cancer metastasis and progression, however, the roles of transcription regulation in angiogenesis are not fully defined. ZNF322A is an oncogenic zinc-finger transcription factor. Here, we demonstrate a new mechanism of Kras mutation-driven ZNF322A transcriptional activation and elucidate the interplay between ZNF322A and its upstream transcriptional regulators and downstream transcriptional targets in promoting neo-angiogenesis. Methods: Luciferase activity, RT-qPCR and ChIP-qPCR assays were used to examine transcription regulation in cell models. In vitro and in vivo angiogenesis assays were conducted. Immunohistochemistry, Kaplan-Meier method and multivariate Cox regression assays were performed to examine the clinical correlation in tumor specimens from lung cancer patients. Results: We validated that Yin Yang 1 (YY1) upregulated ZNF322A expression through targeting its promoter in the context of Kras mutation. Reconstitution experiments by knocking down YY1 under KrasG13V activation decreased KrasG13V-promoted cancer cell migration, proliferation and ZNF322A promoter activity. Knockdown of YY1 or ZNF322A attenuated angiogenesis in vitro and in vivo. Notably, we validated that ZNF322A upregulated the expression of sonic hedgehog (Shh) gene which encodes a secreted factor that activates pro-angiogenic responses in endothelial cells. Clinically, ZNF322A protein expression positively correlated with Shh and CD31, an endothelial cell marker, in 133 lung cancer patient samples determined using immunohistochemistry analysis. Notably, patients with concordantly high expression of ZNF322A, Shh and CD31 correlated with poor prognosis. Conclusions: These findings highlight the mechanism by which dysregulation of Kras/YY1/ZNF322/Shh transcriptional axis enhances neo-angiogenesis and cancer progression in lung cancer. Therapeutic strategies that target Kras/YY1/ZNF322A/Shh signaling axis may provide new insight on targeted therapy for lung cancer patients.

Epigenetic silencing of AATK in acinar to ductal metaplasia in murine model of pancreatic cancer.

BACKGROUND: Cancer subtype switching, which involves unclear cancer cell origin, cell fate decision, and transdifferentiation of cells within a confined tumor microenvironment, remains a major problem in pancreatic cancer (PDA). RESULTS: By analyzing PDA subtypes in The Cancer Genome Atlas, we identified that epigenetic silencing of apoptosis-associated tyrosine kinase (AATK) inversely was correlated with mRNA expression and was enriched in the quasi-mesenchymal cancer subtype. By comparing early mouse pancreatic lesions, the non-invasive regions showed AATK co-expression in cells with acinar-to-ductal metaplasia, nuclear VAV1 localization, and cell cycle suppression; but the invasive lesions conversely revealed diminished AATK expression in those with poorly differentiated histology, cytosolic VAV1 localization, and co-expression of p63 and HNF1α. Transiently activated AATK initiates acinar differentiation into a ductal cell fate to establish apical-basal polarization in acinar-to-ductal metaplasia. Silenced AATK and ectopically expressed p63 and HNF1α allow the proliferation of ductal PanINs in mice. CONCLUSION: Epigenetic silencing of AATK regulates the cellular transdifferentiation, proliferation, and cell cycle progression in converting PDA-subtypes.

AKT-mediated phosphorylation enhances protein stability and transcription activity of ZNF322A to promote lung cancer progression.

ZNF322A is an oncogenic zinc-finger transcription factor. Our published results show that ZNF322A positively regulates transcription of alpha-adducin (ADD1) and cyclin D1 (CCND1) to promote tumorgenicity of lung cancer. However, the upstream regulatory mechanisms of ZNF322A protein function remain elusive. Here, we demonstrate that AKT could phosphorylate ZNF322A by in vitro kinase assay and cell-based mass spectrometry analysis. Overexpression of AKT promoted ZNF322A protein stability and transcriptional activity, whereas these effects were inhibited by knockdown of AKT or treating with AKT inhibitor. We studied AKT-mediated phosphorylation sites, viz. Thr-150, Ser-224, Thr-234, and Thr-262. ZNF322A phosphorylation at Thr-262 by AKT promoted ZNF322A protein stability thus increased ADD1 promoter activity. Interestingly, phosphorylation at Thr-150, Ser-224, and Thr-234 enhanced transcription activity without affecting protein stability of ZNF322A. Chromatin immunoprecipitation and DNA affinity precipitation assays showed that ZNF322A phosphorylation defective mutants Thr-150A, Ser-224A, and Thr-234A attenuated chromatin binding and DNA binding affinity to ADD1 and CCND1 promoters compared with wild-type ZNF322A. Furthermore, AKT-mediated Thr-150, Ser-224, Thr-234, and Thr-262 phosphorylation promoted lung cancer cell growth and metastasis in vitro and in vivo. Clinically, expression of phosphorylated ZNF322A (p-ZNF) correlated with actively phosphorylated AKT (p-AKT) in tumor specimens from 150 lung cancer patients. Multivariate Cox regression analysis indicated that combined p-AKT and p-ZNF expression profile was an independent factor to predict the clinical outcome in lung cancer patients. Our results reveal a new mechanism of AKT signaling in promoting ZNF322A protein stability and transcriptional activity in lung cancer cell, xenograft, and clinical models.

SOX17 overexpression sensitizes chemoradiation response in esophageal cancer by transcriptional down-regulation of DNA repair and damage response genes.

BACKGROUND: Prognosis of esophageal squamous cell carcinoma (ESCC) patients is poor and the concurrent chemoradiation therapy (CCRT) provided to ESCC patients often failed due to resistance. Therefore, development of biomarkers for predicting CCRT response is immensely important. In this study, we evaluated the predicting value of SRY (sex determining region Y)-box 17 (SOX17) protein during CCRT and its dysregulation of transcriptional targets in CCRT resistance in ESCC. METHODS: Pyrosequencing methylation, RT-qPCR and immunohistochemistry assays were performed to examine the DNA methylation, mRNA expression and protein expression levels of SOX17 in endoscopic biopsy from a total of 70 ESCC patients received CCRT. Cell proliferation, clonogenic survival and xenograft growth were used to confirm the sensitization of ESCC cell line KYSE510 in response to cisplatin, radiation or CCRT treatment by SOX17 overexpression in vitro and in vivo. Luciferase activity, RT-qPCR and ChIP-qPCR assays were conducted to examine transcription regulation of SOX17 in KYSE510 parental, KYSE510 radio-resistant cells and their derived xenografts. RESULTS: High DNA methylation coincided with low mRNA and protein expression levels of SOX17 in pre-treatment endoscopic biopsy from ESCC patients with poor CCRT response. SOX17 protein expression exhibited a good prediction performance in discriminating poor CCRT responders from good responder. Overexpression of SOX17 sensitized KYSE510 radio-resistant cells to cisplatin, radiation or CCRT treatment in cell and xenograft models. Importantly, SOX17 transcriptionally down-regulated DNA repair and damage response-related genes including BRCA1, BRCA2, RAD51, KU80 DNAPK, p21, SIRT1, NFAT5 and REV3L in KYSE510 radio-resistant cells to achieve the sensitization effect to anti-cancer treatment. Low expression of BRCA1, DNAPK, p21, RAD51 and SIRT1 was confirmed in SOX17 sensitized xenograft tissues derived from radio-resistant ESCC cells. CONCLUSIONS: Our study reveals a novel mechanism by which SOX17 transcriptionally inactivates DNA repair and damage response-related genes to sensitize ESCC cell or xenograft to CCRT treatment. In addition, we establish a proof-of-concept CCRT prediction biomarker using SOX17 immunohistochemical staining in pre-treatment endoscopic biopsies to identify ESCC patients who are at high risk of CCRT failure and need intensive care.

Rab37 mediates exocytosis of secreted frizzled-related protein 1 to inhibit Wnt signaling and thus suppress lung cancer stemness.

Recent studies have revealed that dysregulated Rab small GTPase-mediated vesicle trafficking pathways are associated with cancer progression. However, whether any of the Rabs plays a suppressor role in cancer stemness is least explored. Rab37 has been postulated as a tumor suppressive small GTPase for trafficking anti-tumor cargos. Here, we report a previously uncharacterized mechanism by which Rab37 mediates exocytosis of secreted frizzled-related protein-1 (SFRP1), an extracellular antagonist of Wnt, to suppress Wnt signaling and cancer stemness in vitro and in vivo. Reconstitution experiments indicate that SFRP1 secretion is crucial for Rab37-mediated cancer stemness suppression and treatment with SRPP1 recombinant protein reduces xenograft tumor initiation ability. Clinical results confirm that concordantly low Rab37, low SFRP1, and high Oct4 stemness protein expression profile can be used as a biomarker to predict poor prognosis in lung cancer patients. Our findings reveal that Rab37-mediated SFRP1 secretion suppresses cancer stemness, and dysregulated Rab37-SFRP1 pathway confers cancer stemness via the activation of Wnt signaling. Rab37-SFRP1-Wnt axis could be a potential therapeutic target for attenuating lung cancer stemness.

Factor VII-Induced MicroRNA-135a Inhibits Autophagy and Is Associated with Poor Prognosis in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common and aggressive malignancies worldwide. Treatment outcomes remain poor mainly due to lack of good diagnostic/prognostic markers and limited therapeutic strategies. We previously characterized aberrant activation of the TF/FVII/PAR2 pathway, which subsequently results in decreased autophagy, as a crucial event in malignant progression of HCC. Here, we identified miR-135a as a highly upregulated miRNA in HCC in response to TF/FVII/PAR2 activation. Analyzing 103 HCC patient specimens, we confirmed that miR-135a was frequently elevated in HCC tissues with higher FVII expression compared to adjacent non-cancerous counterparts. Increased miR-135a levels in HCC were also associated with tumor staging, recurrence, microvascular invasion, and decreased disease-free survival. We subsequently identified Atg14, a key component that regulates the formation of autophagosome as a direct target of miR-135a. Ectopic expression of miR-135a suppressed Atg14 levels and inhibited the autophagic processes. Our results indicate strong positive correlations between miR-135a levels and malignant behaviors in HCC patients and also suggest novel functions of miR-135a in regulation of autophagy, which could be useful as a potential target for prognostic and therapeutic uses.

A six-CpG panel with DNA methylation biomarkers predicting treatment response of chemoradiation in esophageal squamous cell carcinoma.

BACKGROUND: Prognosis of esophageal squamous cell carcinoma (ESCC) patients remains poor, and the chemoradiotherapy (CRT) applied to ESCC patients often failed. Therefore, development of biomarkers to predict CRT response is immensely important for choosing the best treatment strategy of an individual patient. METHODS: The methylation array and pyrosequencing methylation assay were performed in pre-treatment endoscopic biopsies to identify probes with differential CpG methylation levels between good and poor CRT responders in a cohort of 12 ESCC patients. Receiver operating characteristic curves and multivariate logistic regressions were conducted to build the risk score equation of selected CpG probes in another cohort of 91 ESCC patients to predict CRT response. Kaplan-Meier analysis was used to estimate progression-free survival or time-to-progression of patients predicted with good and poor CRT responses. RESULTS: Nine differentially methylated CpG probes were identified to be associated with CRT response. A risk score equation comprising six CpG probes located in IFNGR2, KCNK4, NOTCH4, NPY, PAX6, and SOX17 genes were built. The risk score was derived from the sum of each probe multiplied by its corresponding coefficient. Such a risk score has a good prediction performance in discriminating poor CRT responders from good responders (AUC: 0.930). Moreover, poor CRT responders predicted by risk score significantly had poorer prognosis in terms of shorter progression-free survival and time-to-progression (p = 0.004-0.008). CONCLUSION: We established a proof-of-concept CRT response prediction panel consisting of six-CpG methylation biomarkers in identifying ESCC patients who are at high risk of CRT failure and need intensive care.

A prognostic predictor panel with DNA methylation biomarkers for early-stage lung adenocarcinoma in Asian and Caucasian populations.

BACKGROUND: The incidence of lung adenocarcinoma (LUAD) is increasing worldwide with different prognosis even in early-stage patients. We aimed to identify a prognostic panel with multiple DNA methylation biomarkers to predict survival in early-stage LUAD patients of different racial groups. METHODS: The methylation array, pyrosequencing methylation assay, Cox regression and Kaplan-Meier analyses were conducted to build the risk score equations of selected probes in a training cohort of 69 Asian LUAD patients. The risk score model was verified in another cohort of 299 Caucasian LUAD patients in The Cancer Genome Atlas (TCGA) database. RESULTS: We performed a Cox regression analysis, in which the regression coefficients were obtained for eight probes corresponding to eight genes (AGTRL1, ALDH1A3, BDKRB1, CTSE, EFNA2, NFAM1, SEMA4A and TMEM129). The risk score was derived from sum of each methylated probes multiplied by its corresponding coefficient. Patients with the risk score greater than the median value showed poorer overall survival compared with other patients (p = 0.007). Such a risk score significantly predicted patients showing poor survival in TCGA cohort (p = 0.036). A multivariate analysis was further performed to demonstrate that the eight-probe panel association with poor outcome in early-stage LUAD patients remained significant even after adjusting for different clinical variables including staging parameters (hazard ratio, 2.03; p = 0.039). CONCLUSIONS: We established a proof-of-concept prognostic panel consisting of eight-probe signature to predict survival of early-stage LUAD patients of Asian and Caucasian populations.

Deregulation of SLIT2-mediated Cdc42 activity is associated with esophageal cancer metastasis and poor prognosis.

INTRODUCTION: SLIT2, a secreted protein, has been found to inactivate Cdc42 GTPase to modulate neural cell migration. However, alteration of SLIT2-mediated Cdc42 in terms of migration regulation remains undefined in esophageal squamous cell carcinoma (ESCC). METHODS: We report here in ESCC cell, animal, and clinical models that SLIT2 acts as a migration suppressor and serves as a prognostic biomarker. RESULTS: The immunohistochemistry data indicated that 31.8% (49 of 154) of tumors from ESCC patients showed low expression of SLIT2 protein which correlated with poor overall survival and disease-free survival. DNA methylation analysis suggested that promoter hypermethylation is responsible for low expression of SLIT2 in ESCC. Knockdown of SLIT2 increased ESCC cell migration, while SLIT2 stable overexpression reduced cell migration. ESCC cells treated with conditioned media from cells overexpressing SLIT2 also suppressed cell migration. Importantly, silencing of SLIT2 decreased the complex formation, and thus induced Cdc42 activity and promoted membrane localization of focal adhesion kinase and Paxillin. Anti-metastatic effect of SLIT2 was confirmed in an experimental metastasis model of SLIT2 knockdown ESCC cells. CONCLUSION: Our results provide novel evidence that low expression of SLIT2 correlates with poor prognosis and promotes metastasis in ESCC, which may be regulated by the Cdc42-mediated pathways.

Small GTPase Rab37 targets tissue inhibitor of metalloproteinase 1 for exocytosis and thus suppresses tumour metastasis.

Rab small GTPases are master regulators of membrane trafficking and guide vesicle targeting. Recent publications show that Rab-controlled trafficking pathways are altered during tumorigenesis. However, whether any of the Rabs plays a metastasis suppressor role is least explored. Here we address the metastasis suppressive function of human Rab37 (hRAB37) using secretomics, cell, animal and clinical analyses. We show that tissue inhibitor of metalloproteinase 1 (TIMP1), a secreted glycoprotein that inhibits extracellular matrix turnover, is a novel cargo of hRAB37. hRAB37 regulates the exocytosis of TIMP1 in a nucleotide-dependent manner to inactivate matrix metalloproteinase 9 (MMP9) migration axis in vitro and in vivo. Dysfunction of hRAB37 or TIMP1 abrogates metastasis suppression. Lung cancer patients with metastasis and poor survival show low hRAB37 protein expression coinciding with low TIMP1 in tumours. Our findings identify hRAB37 as a novel metastasis suppressor Rab that functions through the TIMP1-MMP9 pathway and has significant prognostic power.

Network-based analysis identifies epigenetic biomarkers of esophageal squamous cell carcinoma progression.

MOTIVATION: A rapid progression of esophageal squamous cell carcinoma (ESCC) causes a high mortality rate because of the propensity for metastasis driven by genetic and epigenetic alterations. The identification of prognostic biomarkers would help prevent or control metastatic progression. Expression analyses have been used to find such markers, but do not always validate in separate cohorts. Epigenetic marks, such as DNA methylation, are a potential source of more reliable and stable biomarkers. Importantly, the integration of both expression and epigenetic alterations is more likely to identify relevant biomarkers. RESULTS: We present a new analysis framework, using ESCC progression-associated gene regulatory network (GRN escc), to identify differentially methylated CpG sites prognostic of ESCC progression. From the CpG loci differentially methylated in 50 tumor-normal pairs, we selected 44 CpG loci most highly associated with survival and located in the promoters of genes more likely to belong to GRN escc. Using an independent ESCC cohort, we confirmed that 8/10 of CpG loci in the promoter of GRN escc genes significantly correlated with patient survival. In contrast, 0/10 CpG loci in the promoter genes outside the GRN escc were correlated with patient survival. We further characterized the GRN escc network topology and observed that the genes with methylated CpG loci associated with survival deviated from the center of mass and were less likely to be hubs in the GRN escc. We postulate that our analysis framework improves the identification of bona fide prognostic biomarkers from DNA methylation studies, especially with partial genome coverage.