m6A methyltransferase METTL16 mediates immune evasion of colorectal cancer cells via epigenetically regulating PD-L1 expression.

BACKGROUND: The immune checkpoint inhibitors (ICIs) has dramatically changed the therapeutic area of cancers. A great number of patients with CRC exhibit poor response rate to ICI treatment. N6-methyl adenosine (m6A) is closely correlated with the initiation and progression of cancers. To explore the role of methyltransferase-like 16 (METTL16) in CRC treatment. METHODS: Clinical samples and different CRC cell lines were collected. The expression of METTL16 and PD-L1 was determined by qPCR, IHC. Ectopic expression of METTL16 was performed in CRC cells. A co-culture system was established using CRC cells and T cells to measure the immune evasion. Cell viability, apoptosis, migration, and invasion were examined by CCK-8, colony formation, flow cytometry, Transwell, and wound healing assay, respectively. The N6-methyl adenosine (m6A) modification of PD-L1 by METTL16 was investigated by methylated RIP (MeRIP) and RNA stability experiment. In vivo xenograft model was established to measure the effects of METTL16 on CRC growth. RESULTS: METTL16 was decreased and PD-L1 was increased in CRC tissues and cell lines. METTL16 enhanced cell proliferation, migration, and invasion, and promoted CRC tumor growth in vivo. METTL16 induced m6A modification and decreased the stability of METTL16 RNA, leading to the suppression of METTL16 level. METTL16 overexpression in CRC cells induced decreased portion of PD-1 positive T cells. Overexpression of METTL16 and inhibition of PD-1 synergistically suppressed in vivo growth of CRC cells. CONCLUSIONS: Our work identified the METTL16/PD-L1/PD-1 regulatory axis in CRC development and immune evasion, which represented a promising target for CRC treatment.

LncRNA AC093818.1 accelerates gastric cancer metastasis by epigenetically promoting PDK1 expression.

Gastric cancer (GC) is a highly prevalent type of metastatic tumor. The mechanisms underlying GC metastasis are poorly understood. Some long noncoding RNAs (lncRNAs) reportedly play key roles in regulating metastasis of GC. However, the biological roles of five natural antisense lncRNAs (AC093818.1, CTD-2541M15.1, BC047644, RP11-597M12.1, and RP11-40A13.1) in GC metastasis remain unclear. In this study, the expression of these lncRNAs was measured by quantitative reverse transcription-polymerase chain reaction. Migration and invasion were evaluated by wound-healing and the Transwell assay, respectively. Stable cells were injected into the tail veins of nude mice. Sections of collected lung and liver tissues were stained using hematoxylin and eosin. Protein expression was analyzed by western blot. RNA immunoprecipitation (RIP) assay was used to verify whether the STAT3 and SP1 transcription factors bound to AC093818.1 in GC cells. Expression levels of the five lncRNAs, especially AC093818.1, were significantly upregulated in metastatic GC tissues relative to those in nonmetastatic GC tissues. AC093818.1 expression was correlated with invasion, lymphatic metastasis, distal metastasis, and tumor-node-metastasis stage. AC093818.1 expression was highly sensitive and specific in the diagnosis of metastatic or nonmetastatic GC. AC093818.1 overexpression promoted GC migration and invasion in vitro and in vivo. AC093818.1 overexpression increased PDK1, p-AKT1, and p-mTOR expression levels. AC093818.1 silencing decreased these expressions. AC093818.1 bound to transcription factors STAT3 and SP1, and SP1 or STAT3 silencing could alleviated the effect of AC093818.1 overexpression. The data demonstrate that lncRNA AC093818.1 accelerates gastric cancer metastasis by epigenetically promoting PDK1 expression. LncRNA AC093818.1 may be a potential therapeutic target for metastatic GC.

Ascites do not affect the rate of complete cytoreductive surgery and prognosis in patients with primary ovarian cancer with ascites treated with hyperthermic intraperitoneal chemotherapy.

Cytoreductive surgery (CRS) is the current standard therapy procedure for patients with advanced ovarian cancer (OC), but numerous patients with OC are complicated with ascites. The aim of the present study was to assess whether massive ascites affect the rate of complete CRS and prognosis for patients with primary OC treated with hyperthermic intraperitoneal chemotherapy (HIPEC). Between December 2006 and December 2015, 1,293 patients with primary OC from the Intracelom Hyperthermic Perfusion Therapy Center of the Cancer Hospital of Guangzhou Medical University prospective database were treated with CRS combined with HIPEC. A total of 1,225 patients were without malignant ascites or small amounts of ascites and 68 had massive malignant ascites. The rate of complete CRS, overall survival (OS), disease-free survival (DFS) and resolution of ascites for patients with massive ascites were analyzed between patients without/small ascites, and with massive ascites. Complete CRS was successful in 86.8% (1,063/1,225) of patients without/small ascites, and 85.3% (58/68) of patients with massive ascites. No statistical differences were identified in complete CRS success between patients with ascites and patients without/small ascites (P=0.080). For patients with massive ascites, all symptoms exhibited regression; the total objective remission rate was 100% (68/68), even for patients with incomplete CRS (10/68) (P=0.100). The mean OS was 58 months and the mean DFS was 26 months in patients without/small ascite, vs. 57 months and 28 months in patients with massive ascites. No significant differences were noted in median DFS and median OS between patients with ascites, and patients without/small ascites (All P>0.05). In conclusion, the results of the present study suggest that ascites does not affect the rate of complete CRS and the prognosis of patients with massive ascites following HIPEC. CRS is suitable for the majority of patients with primary OC and massive ascites.

Hyperthermia enhances radiosensitivity of colorectal cancer cells through ROS inducing autophagic cell death.

Hyperthermia (HT) enhances the anti-cancer effects of radiotherapy (RT), but the precise biochemical mechanisms involved are unclear. This study was aim to investigate if mild HT sensitizes colorectal cancer cells to RT through reactive oxygen species (ROS)-inducing autophagic cell death in a mice model of HCT116 human colorectal cancer. HCT116 mice model were randomly divided into five groups: mock group, hyperthermia group (HT), radiotherapy group (RT), HT + RT group, and HT + RT +N-acetyl L-cysteine (NAC) group (HT + CT + NAC). After four weeks of treatment, cancer growth inhibition, rate and mitochondrial membrane potential were measured with MTT and JC-1 assays, respectively, while ROS were estimated fluorimetrically. The relationship of these parameters to expressions of autophagy-related genes Beclin1, LC3B, and mTOR was analyzed. Gene expression was measured by Real-Time polymerase chain reaction (RT-PCR). There were significant increases in ROS levels and mitochondrial membrane potential in the HT + RT group. ROS levels in the HT + RT group increased more significantly than in any other group. In contrast, ROS levels in the HT + RT + NAC group were significantly decreased relative to the HT + RT group. The number of autophagic bodies in HT + RT group was higher than that of mock group. There were significant increases in the expression of Beclin1 and LC3B genes, while mTOR expression was significantly decreased in the HT + CT group. Treatment with NAC reversed the pattern of these changes. These results indicate that HT enhances the radiosensitivity of colorectal cancer cells to RT through ROS inducing autophagic cell death.

BRD4 promotes gastric cancer progression through the transcriptional and epigenetic regulation of c-MYC.

Although the significance of BRD4 in the epigenetic memory and cancer genesis has been intensively investigated, little is known about its function and potential roles during the generation and progression of gastric cancer. We report here that BRD4 increases the proliferation and represses the apoptosis of gastric cancer cells through activating c-MYC via transcriptional and epigenetic regulation mechanisms. Expression analyses in both small and large cohort of sample show that BRD4 is highly expressed in gastric cancer tissues/cells when compared with the adjacent non-tumor tissues/normal cells. We also find a positive correlation between the expression of BRD4 and c-MYC in patient samples. The repression of BRD4 by siRNAs leads to the down-regulation of c-MYC in gastric cancer cells. Chromatin immunoprecipitation-qPCR and luciferase assays show that BRD4 binds to and coordinately activates c-MYC promoter, indicating that c-MYC is transcriptional target of BRD4 and BRD4 regulates its basal expression. Further evidence show that the histone acetylation inhibitor reduces the binding of BRD4 as well as the histone activation level on c-MYC promoter, and leads to the down-regulation of c-MYC, suggesting that BRD4 regulates the expression of c-MYC through epigenetic mechanism. Functionally, the suppression of BRD4 leads to growth inhibition and apoptosis in gastric cancer cells. Force expression of c-MYC alongside with BRD4 repression rescue the anti-cancer effects caused by BRD4 repression. Collectively, our data not only uncovered the mechanism of BRD4 in regulating the proliferation of gastric cancer cells but also provides a new therapeutic strategy for this type of cancer.

Long noncoding RNA LINC00673 epigenetically suppresses KLF4 by interacting with EZH2 and DNMT1 in gastric cancer.

Long non-coding RNAs (lncRNAs), a variety of transcripts without protein coding ability, have recently been reported to play vital roles in gastric cancer (GC) development and progression. However, the biological role of long non-coding RNA LINC00673 in GC is not fully known. In the study, we found that LINC00673 expression was dramatically higher in gastric cancer tissues compared with adjacent normal tissues, and positively associated with lymph node metastasis, distant metastasis and TNM stage in patients. Higher LINC00673 expression predicted poor disease-free survival (DFS) and overall survival (OS) in GC patients. By univariate and multivariate Cox analysis, the results confirmed that higher LINC00673 expression was an independent risk factor of prognosis in patients. Knockdown of endogenous LINC00673 significantly inhibited cell proliferation, colony formation number, cell migration and invasion in GC. Furthermore, knockdown of endogenous LINC00673 reduced the expression levels of PCNA, CyclinD1 and CDK2 in GC cells. RNA immunoprecipitation (RIP) and chromatin immunoprecipitation (ChIP) proved that LINC00673 suppressed KLF4 expression by interacting with EZH2 and DNMT1 in GC cells. Moreover, we confirmed that LINC00673 promoted cell proliferation and invasion by partly repressing KLF4 expression in GC. Taken together, these results indicated that LINC00673 may be a prognostic biomarker and therapeutic target for GC patients.

Mild hyperthermia enhances sensitivity of gastric cancer cells to chemotherapy through reactive oxygen species-induced autophagic death.

Mild hyperthermia enhances anti-cancer effects of chemotherapy, but the precise biochemical mechanisms involved are not clear. This study was carried out to investigate whether mild hyperthermia sensitizes gastric cancer cells to chemotherapy through reactive oxygen species-induced autophagic death. In total, 20 BABL/c mice of MKN-45 human gastric cancer tumor model were divided into hyperthermia + chemotherapy group, hyperthermia group, chemotherapy group, N-acetyl-L-cysteine group, and mock group. Reactive oxygen species production and expression of autophagy-related genes Beclin1, LC3B, and mammalian target of rapamycin were determined. The relationships between tumor growth regression, expression of autophagy-related genes, and reactive oxygen species production were evaluated. Tumor size and wet weight of hyperthermia + chemotherapy group was significantly decreased relative to values from hyperthermia group, chemotherapy group, N-acetyl-L-cysteine group, and mock group ( F = 6.92, p < 0.01 and F = 5.36, p < 0.01, respectively). Reactive oxygen species production was significantly higher in hyperthermia + chemotherapy group than in hyperthermia, chemotherapy, and mock groups. The expression levels of Beclin1 and LC3B were significantly higher, while those of mammalian target of rapamycin were significantly lower in hyperthermia + chemotherapy group than in hyperthermia, chemotherapy, and mock groups. Tumor growth regression was consistent with changes in reactive oxygen species production and expression of autophagy-related genes. N-acetyl-L-cysteine inhibited changes in the expression of the autophagy-related genes and also suppressed reactive oxygen species production and tumor growth. Hyperthermia + chemotherapy increase expression of autophagy-related genes Beclin1 and LC3B, decrease expression of mammalian target of rapamycin, and concomitantly increase reactive oxygen species generation. These results strongly indicate that mild hyperthermia enhances sensitivity of gastric cancer cells to chemotherapy through reactive oxygen species-induced autophagic death.

Bladder intracavitary hyperthermic perfusion chemotherapy for the prevention of recurrence of non-muscle invasive bladder cancer after transurethral resection.

Preventing the recurrence of non-muscle invasive bladder cancer (NMIBC) post-transurethral resection (TUR) remains challenging. The aim of the present study was to investigate the effectiveness and safety of bladder intracavitary hyperthermic perfusion chemotherapy (BHPC) for prevention of NMIBC recurrence post-TUR. Between December 2006 and December 2014, 53 patients with NMIBC who underwent TUR were randomly assigned to receive BHPC (BHPC group, 28 patients) or intravesical chemotherapy alone (chemotherapy group, 25 patients) at the Intracelom Hyperthermic Perfusion Therapy Center of Guangzhou Medical University Cancer Hospital (Guangzhou, China). BHPC was performed by combining perfusion-based hyperthermia with chemotherapeutic agent mitomycin C (MMC) in the bladder, and the chemotherapy group of patients received bladder MMC perfusion. The concentration of MMC in the perfusion fluid and serum were assessed at different time-points. Tumor recurrence, disease-free survival (DFS), and side-effects were recorded and compared between the 2 groups. Results revealed that BHPC was performed smoothly, at ~44̊C in the bladder cavity. Patients tolerated BHPC, and no side-effects were observed. Both BHPC and intravesical chemotherapy achieved a high MMC concentration in the bladder perfusion liquid, but low MMC concentration in the serum, although serum MMC concentrations in the BHPC group were significantly higher (P<0.05). The tumor recurrence rate was significantly lower (10.7 vs. 28.0%; P=0.02) and the DFS period was significantly longer (37±1.2 vs. 19±0.9 months; P=0.001) in the BHPC group than in the chemotherapy group. Our results demonstrated that BHPC is safe and effective for preventing NMIBC recurrence post-TUR and prolongs DFS.

Hyperthermic Intraperitoneal Perfusion Chemotherapy and Cytoreductive Surgery for Controlling Malignant Ascites From Ovarian Cancer.

BACKGROUND: Malignant ascites, a complication often seen in patients with ovarian cancer (OC), is difficult to treat, but hyperthermic intraperitoneal chemotherapy (HIPEC) has a good efficacy. OBJECTIVE: The aim of this study was to assess the efficacy of cytoreductive surgery (CRS) combined with HIPEC for controlling malignant ascites from OC. MATERIALS AND METHODS: From December 2009 until December 2014, 53 patients with OC and malignant ascites were treated with CRS and HIPEC. Patients in good health condition were treated with CRS followed by HIPEC (CRS + HIPEC), and patients in poor health condition were treated initially with B-mode ultrasound-guided HIPEC followed by delayed CRS upon improvement of their health condition (HIPEC + delayed CRS). Resolution of ascites, complete CRS, overall survival, and disease-free survival were analyzed. RESULTS: All patients showed ascites regression. The total objective remission rate was 100%, even for patients in the poor condition group before CRS. Complete CRS was successful in 30 (88.23%) of 34 patients in the good condition group, and 17 (89.47%) of 19 patients in the poor condition group (P > 0.05). Median disease-free survival and median overall survival were 21 and 39 months in the good condition group, and 22 and 38 months in the poor condition group, respectively (P > 0.05). CONCLUSIONS: Hyperthermic intraperitoneal chemotherapy is effective at controlling ascites in patients with OC, even for patients in poor condition before CRS, or when complete CRS is not feasible. Furthermore, the regression of ascites appears not to be dependent on complete resection.