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Several recent studies suggested that TLKs are related to tumor progression. However, the function and action mechanism of TLK2 in GC remain elusive. We observed that TLK2 was significantly upregulated in GC and acted as an independent prognostic factor in GC patients. In line with these results, TLK2 silencing markedly reduced GC aggressiveness, whereas its overexpression had the opposite effect. IP-MS suggested that the effect of TLK2 expression on GC was mainly associated with metabolism reprogramming. TLK2 knockdown suppressed amino synthesis through downregulating the mTORC1 pathway and ASNS expression in GC. Mechanistically, mTORC1 directly interacted with ASNS and inhibited its protein degradation. Further experiments confirmed that ASNS protein degradation was ubiquitination degradation rather than degradation through autophagy. Inhibiting and activating the mTORC1 pathway can upregulate and downregulate ASNS ubiquitination, respectively. Moreover, the mTORC1 pathway can reverse the protein regulation of ASNS by TLK2. Notably, TLK2 regulated the ASNS mRNA levels. Further evidence confirmed that TLK2 directly interacted with ATF4, a transcription factor of ASNS, and promoted the expression of ATF4. Finally, we found kinase inhibitor Fostamatinib can significantly inhibit the proliferation, invasion, and migration of GC cells by inhibiting TLK2 enzyme activity. Taken together, this study reveals a novel functional link between TLK2, the mTORC1/ASNS axis, and GC. Therefore, TLK2 could be a potential therapeutic approach for GC target therapy.

The poor efficacy of chimeric antigen receptor T-cell therapy (CAR T) for solid tumor is due to insufficient CAR T cell tumor infiltration, in vivo expansion, persistence, and effector function, as well as exhaustion, intrinsic target antigen heterogeneity or antigen loss of target cancer cells, and immunosuppressive tumor microenvironment (TME). Here we describe a broadly applicable nongenetic approach that simultaneously addresses the multiple challenges of CAR T as a therapy for solid tumors. The approach massively reprograms CAR T cells by exposing them to stressed target cancer cells which have been exposed to the cell stress inducer disulfiram (DSF) and copper (Cu)(DSF/Cu) plus ionizing irradiation (IR). The reprogrammed CAR T cells acquired early memory-like characteristics, potent cytotoxicity, enhanced in vivo expansion, persistence, and decreased exhaustion. Tumors stressed by DSF/Cu and IR also reprogrammed and reversed immunosuppressive TME in humanized mice. The reprogrammed CAR T cells, derived from peripheral blood mononuclear cells (PBMC) of healthy or metastatic breast cancer patients, induced robust, sustained memory and curative anti-solid tumor responses in multiple xenograft mouse models, establishing proof of concept for empowering CAR T by stressing tumor as a novel therapy for solid tumor.

This study evaluated the impact of a new intracorporeal π-shaped esophagojejunostomy (EJS) and double-tract reconstruction (DTR) in totally laparoscopic and totally robotic proximal gastrectomy (TLPG or TRPG) for treating upper third early gastric cancer (U-EGC) in terms of intraoperative and short-term postoperative outcomes. Early proximal gastric cancer patients were identified based on a prospectively established database. From January 2017 to December 2018, these patients underwent intracorporeal π-shaped EJS and DTR after totally laparoscopic (n = 8) or robotic (n = 4) proximal gastrectomy (PG). We recorded and analyzed the baseline characteristics and surgical outcomes, including postoperative complications for these patients. No severe postoperative complications were observed following the operational procedures. Twelve patients (seven male and five female) diagnosed with cardia cancer (Siewert II and III) were enrolled, of which eight underwent the totally laparoscopic proximal gastrectomy (TLPG), and four underwent the totally robotic proximal gastrectomy (TRPG). The mean operative time, blood loss, day of the start of the diet, and postoperative hospital stay was 235.54 ± 20.79 min, 50.65 ± 35.44 mL, 3.85 ± 0.65 days, and 12.45 ± 3.24 days, respectively. All patients presented with a diagnosis of stage I gastric cancer. The mean number of lymph node dissections and the maximum tumor diameter was 13.91 ± 4.63 and 2.18 ± 0.73 cm, respectively. After the operational procedure, using the iodoethylene contrast reagent, we observed that a large proportion of iodoethylene contrast agents entered the jejunum directly, and a small proportion entered the jejunum through the duodenum. Surgeons followed up with ten patients for more than 12 months and the remaining two patients for more than 24 months. None of the patients showed any signs of anastomotic stenosis or reflux esophagitis or anemia symptoms. This study presents a novel method for π-shaped EJS and DTR as an alternative in TLPG or TRPG for treating proximal early gastric cancer, and it offers better short-term postoperative and intraoperative surgical outcomes.

Purpose: Hypoxia-inducible factor-1alpha (HIF-1A) is a transcription factor that plays an “angiogenic switch” role especially under hypoxia microenvironment in solid tumor. However, the functions and clinical significance of HIF-1A in gallbladder cancer (GBC) are still controversial, and it has not been studied in normal gallbladder tissues. In this study, we sought to clarify the role of sub-cellular localization of HIF-1A expression in GBC and normal gallbladder tissues. Methods: The expressions of HIF-1A and CD34 in 127 GBC and 47 normal gallbladder tissues were evaluated by immunohistochemistry. Cox's proportional hazards model analysis and Kaplan-Meier method analysis were used to assess the correlations between these factors and clinicopathological features and prognosis. Results: HIF-1A was expressed in both cytoplasm and nucleus of GBC and normal control tissues, and was significantly correlated with microvessel density (MVD). GBC tissues with positive nuclear HIF-1A expression had higher MVD compared to that with positive cytoplasmic HIF-1A expression; however, in normal gallbladder tissues, samples with positive cytoplasmic HIF-1A had higher MVD compared to that with positive nuclear HIF-1A expression. Moreover, GBC with nuclear HIF-1A expression tended to be more poorly differentiated and had larger tumor size compared to that with cytoplasm HIF-1A expression. Furthermore, GBC patients with nuclear HIF-1A positive were significantly correlated with worse overall survival (OS) compared with cytoplasmic HIF-1A positive. Multivariate Cox regression analysis identified lymph node metastasis and nuclear HIF-1A expression to be independent prognostic parameter in GBC. Conclusions: Our findings provide evidence for the first time that HIF-1A is expressed in normal gallbladder tissues. Nuclear HIF-1A and cytoplasm HIF-1A plays different roles in GBC and normal gallbladder tissues.

Radiotherapy (RT) is a key treatment for prostate cancer. However, RT resistance can contribute to treatment failure. Prostate cancer stem cells (PCSCs) are radioresistant. We recently found that fractionated irradiation (FIR) upregulates expression of the immune checkpoint B7-H3 (CD276) on PCSCs and bulk cells in each prostate cancer cell line tested. These findings prompted us to investigate whether B7-H3 targeting chimeric antigen receptor (CAR) T cells, which may abrogate function of an immune checkpoint and mediate lysis of targeted cells, can target RT-resistant PCSCs in vitro and in vivo. B7-H3 expression is naturally higher on PCSCs than bulk prostate cancer cells and cytotoxicity of B7-H3 CAR T cells to PCSCs is more potent than to bulk prostate cancer cells. Furthermore, FIR significantly upregulates B7-H3 expression on PCSCs and bulk prostate cancer cells. The duration of FIR or single-dose irradiation-induced further upregulation of B7-H3 on bulk prostate cancer cells and PCSCs lasts for up to 3 days. B7-H3 CAR T-cell cytotoxicity against FIR-resistant PCSCs at a low effector to target ratio of 1:1 was assessed by flow cytometry and sphere formation assays. Further upregulation of B7-H3 expression by FIR made PCSCs even more sensitive to B7-H3 CAR T-cell–mediated killing. Consequently, the FIR and B7-H3 CAR T-cell therapy combination is much more effective than FIR or CAR T cells alone in growth inhibition of hormone-insensitive prostate cancer xenografts in immunodeficient mice. Our work provides a sound basis for further development of this unique combinatorial model of RT and B7-H3 CAR T-cell therapy for prostate cancer. Significance: We demonstrate that FIR significantly upregulates B7-H3 expression by RT-resistant PCSCs and bulk cells; cytotoxicity of B7-H3 CAR T cells to FIR-treated PCSCs is potent and results in significantly improved antitumor efficacy in mice.

NEDD4L (neural precursor cell expressed developmentally down-regulated 4-like) protein is a member of ubiquitin ligases Nedd4 family. Although studies have shown that Nedd4L may act as a tumor suppressor in various cancers, including gastric cancer (GC), its clinical significance and the diagnostic value in GC is not well defined. HIF-1α (hypoxia inducible factor family of transcription factors) is actively involved in the metabolism of many tumors, although the relationship between its expression levels and clinical significance in GC still need to be established. In this study, the level of HIF-1α and NEDD4L mRNA and protein in 25 freshly frozen GC- and matched normal-tissues were determined by western blot and quantitative PCR (qPCR). Additionally, immunohistochemistry assay was performed to measure the protein level of NEDD4L and HIF-1α in 124 GC and 25 normal control tissues. We observed that the NEDD4L mRNA and protein levels decreased significantly (P < 0.001) in GC tissues, while that of HIF-1α increased (P < 0.001), and they both were associated with a poor prognosis, as was the case in patients with lower NEDD4L and higher HIF-1α expression (P < 0.001). On correlation analysis, a significantly negative relationship (r = 0.288, P < 0.01) was revealed between NEDD4L and HIF-1α expressions. Multivariate analysis revealed that co-expression of NEDD4L (P < 0.05) and HIF-1α (P < 0.001) were independent predictors of GC prognosis. Thus, the correlation of NEDD4L and HIF-1α levels may act as a prognostic marker of GC.

Yi Sheng,1,* Wei Wang,2,* Bo Hong,2 Xingwang Jiang,1 Ruochuan Sun,1 Qiang Yan,1 Shangxin Zhang,1 Mingdian Lu,1 Shengyi Wang,1 Zhen Zhang,1 Wenchu Lin,2 Yongxiang Li1 1Department of General Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China; 2High Magnetic Field Laboratory, Chinese Academy of Sciences, Hefei, Anhui, China *These authors contributed equally to this work Background: KIF20A is well known as one of the key proteins in mitosis. Recently, a number of studies illustrated that KIF20A might function as an oncogene in some carcinomas. However, its expression levels and clinical value remained unclear in gastric cancer (GC). Patients and methods: In this study, we investigated the expression of KIF20A in samples from GC patients and cell lines by quantitative real-time PCR and Western blot. The function of KIF20A in cell proliferation of GC cell lines was examined via cell viability and colony formation assays. Immunohistochemistry assay based on a tissue microarray consisting of 146 cases was performed to evaluate the prognostic value of KIF20A. The overall survival rate of 122 GC patients based on KIF20A expression was analyzed as well. Finally, using KIF20A inhibitor, genistein, and combining it with cisplatin or fluorouracil, the antitumor effects were studied. Results: Most GC samples (56.76%) showed higher KIF20A expression level compared to their corresponding normal specimens, which demonstrated the potential oncogenic role of KIF20A in GC. The functional studies elucidated the essential role of KIF20A in GC cell proliferation. Besides, tissue microarray result showed that the expression level of KIF20A was significantly related to the histological grades (P=0.036). Furthermore, we found the expression of KIF20A was related to poor overall survival rate, which is coincident with the results from Kaplan–Meier plotter database. In addition, we found that a KIF20A inhibitor, genistein, could enhance the antitumor activity of cisplatin and fluorouracil, which might be considered as a chemosensitive agent in GC. Conclusion: KIF20A can promote cell proliferation in GC, which might be used as an independent prognostic factor and a potential therapeutic target. Keywords: KIF20A, genistein, gastric cancer, prognosis

Ting Li, Ruochuan Sun, Mingdian Lu, Jiacong Chang, Xiangling Meng,* Huo Wu* Department of General Surgery, The First Affiliated Hospital of Anhui Medical University, He Fei, 230222, China *These authors contributed equally to this work Background: NDRG3 is an N-myc downregulated gene (NDRG). The aim of this article was to identify the role of NDRG3 in colorectal cancer (CRC) and to determine the mechanism underlying its function. Methods: Using immunohistochemical staining, expression and clinicopathological variables of NDRG3 were analyzed in 170 CRC samples. Overexpression of NDRG3 was employed in SW1116 cells, downregulation of NDRG3 was achieved in RKO cells, then migration and invasion assays were performed in vitro, and a mouse model was constructed in vivo. Results: Increased expression of NDRG3 was observed in primary CRC tissues, and this expression was correlated with distant metastasis. Consistently, ectopic expression of NDRG3 in SW1116 cells enhanced cell migration and invasion, while knockdown of NDRG3 in RKO cells significantly suppressed CRC cell metastasis. The portal vein injection models suggested that NDRG3 overexpression facilitates liver metastasis. These events were associated with the phosphorylation of Src (c-Src) at Tyr 419 site. Conclusion: Our results showed that NDRG3 facilitates CRC migration and invasion by activating Src phosphorylation, suggesting the role of NDRG3 as a candidate oncogene. Keywords: NDRG3, colorectal cancer, metastasis, Src

To explore the expression of KIF18A gene protein in gastric cancer tissues and its association with the prognosis of patients.Twenty fresh paired gastric cancer specimens and adjacent normal mucosa(at least 5 cm from the edge of tumor) from 20 gastric cancer patients undergoing operation in Department of General Surgery at the First Affiliated Hosptial of Anhui Medical University between March 2015 and July 2015 were collected. Real-time PCR was used to examine KIF18A mRNA expression in above specimens. Meanwhile, paraffin embedded cancer tissue samples from 129 gastric cancer patients undergoing operation and 23 samples of randomly selected normal gastric tissue(adjacent non-cancer tissue) were collected to establish the microarray. Immunohistochemistry method was applied to detect the KIF18A protein expression in the microarray after confirmation by pathologists. Association of KIF18A expression with clinicopathological features in gastric cancer patients was evaluated. Cox proportional hazard model was used to identify prognostic risk factors.Among 20 fresh paired gastric cancer specimens, mRNA expression of KIF18A in 16 specimens was obviously lower than that in adjacent normal tissues. The positive rate of KIF18A protein expression in gastric cancer tissues was significantly lower than that in normal gastric tissues in microarray[45.0%(58/129) vs. 69.6%(16/23), P=0.041]. KIF18A protein expression was significantly associated with invasion depth (P=0.008) and TNM staging (P=0.032). The median overall survival of all the 129 patients was 44.0(95% CI: 39.78-49.24) months. The three-year survival rates of patients with high and low KIF18A expression were 67.2% and 36.6% respectively(P=0.020). Cox regression analysis showed that KIF18A expression was an independent protective factor of the prognosis of gastric cancer patients (HR=0.570, 95% CI:0.335 to 0.970).KIF18A expression is down-regulated in gastric cancer tissue, which may play a critical role in gastric cancer carcinogenesis. Lower expression of KIF18A is associated with poor prognosis of gastric cancer patients. KIF18A may be a potential prognostic marker of gastric cancer.

// Youliang Wu 1, * , Dapeng Yun 2, * , Yingjie Zhao 2 , Yuqi Wang 2 , Ruochuan Sun 1 , Qiang Yan 1 , Shangxin Zhang 1 , Mingdian Lu 1 , Zhen Zhang 1 , Daru Lu 2 , Yongxiang Li 1 1 Department of General Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei, People’s Republic of China 2 State Key Laboratory of Genetic Engineering and MOE Key Laboratory of Contemporary Anthropology, School of Life Sciences, Fudan University, Shanghai, People’s Republic of China * These authors have contributed equally to this work Correspondence to: Daru Lu, email: drlu@fudan.edu.cn Yongxiang Li, email: yongxiangli_1@hotmail.com Keywords: gastric cancer, RBMS3, HIF1A, MVD, prognostic marker Received: March 30, 2016 Accepted: November 11, 2016 Published: November 25, 2016 ABSTRACT Frequent loss of multiple regions in short arm of chromosome 3 is found in various tumors including gastric cancer (GC). RNA binding motif, single-stranded interacting protein 3 (RBMS3) is a tumor suppressor gene located in this region and mediates cancer angiogenesis. However, the role of RBMS3 in GC remains unclear. To evaluate whether RBMS3, together with HIF1A, another key regulator of angiogenesis, predicts GC prognosis, the levels of RBMS3 and HIF1A were first examined by quantitative PCR (qPCR) and western blot from 27 fresh frozen GC and paired normal gastric tissues and then tested by immunohistochemistry (IHC) from 191 GC and 46 normal controls. Moreover, uni- and multivariate analysis were employed to assess the correlations between their levels and microvessel density (MVD) and clinical prognosis. To further identify RBMS3 function in vitro , cell proliferation assay, clonogenic assay, flow cytometry analysis and endothelial cell tube formation assay were employed. We found that RBMS3 level was decreased, whereas HIF1A was elevated in GC. Furthermore, we demonstrated that RBMS3 was an independent prognostic factor and the levels of RBMS3 and HIF1A were associated with GC angiogenesis and histopathological differentiation: patients with lower RBMS3 level and higher nuclear HIF1A expression had poorer prognosis. Besides, gain- and loss-of-function study revealed RBMS3 regulation on G1/S progression, cell proliferation and the tube formation of human umbilical vein endothelial cells (HUVECs) in vitro. These findings implicated that RBMS3 and nuclear HIF1A could act as prognostic biomarkers and therapeutic targets for GC.

As one of the most notorious of malignancies, glioblastoma multiforme (GBM), accounts for 45.2% of all malignant primary brain tumors.1 Despite aggressive surgery, radiation, and chemotherapy, the life expectancy of patients averages a mere 14 months after diagnosis.2 Soluble N-ethylmaleimide-sensitive factor (NSF) receptor (SNARE) is a superfamily of small proteins with more than 35 members in mammals, varying in size and primary structure.3 As an essential mechanism for cellular activities, SNARE has been observed in the progression of various tumors.4,5 Vesicle-associated membrane protein 8 (VAMP8) was first identified as an endosomal SNARE that participates in diverse biological functions including endosomal fusion,6–8 the exocytosis of GLUT4 and insulin,9,10 regulation of exocytosis in secretory cells,11 sequential granule-to-granule fusion,11 and autophagy.12,13 Nevertheless, nobody has reported its involvement in tumor progression. Temozolomide (TMZ) is the most commonly used chemotherapeutic drug in clinical trials for glioma. However, a large number of patients are resistant to TMZ, which greatly compromise the clinical treatment. Thus, research on TMZ resistance is of great importance for ameliorating the therapeutic efficacy and alleviating the suffering of patients. Autophagy is a dynamic process of protein degradation that is essential for survival, differentiation, development, and homeostasis.14 Despite evidence demonstrating the role of autophagy in modifying TMZ resistance in GBM,15–17 it remains incompletely understood, and clarification is needed. In this study, we first analyzed the expression and prognostic value of VAMP8 in The Cancer Genome Atlas (TCGA) and a cohort of 282 glioma patients, and then investigated its roles on tumorigenisis and TMZ resistance in glioma. Our findings revealed that the dysregulation of VAMP8 is a potential component of glioma pathogenesis and TMZ resistance, which might become a new therapeutic target for patients with glioma.