The Effect of the Histone Chaperones HSPA8 and DEK on Tumor Immunity in Hepatocellular Carcinoma.

Complex immune contexture leads to resistance to immunotherapy in hepatocellular carcinoma (HCC), and the need for new potential biomarkers of immunotherapy in HCC is urgent. Histone chaperones are vital determinants of gene expression and genome stability that regulate tumor development. This study aimed to investigate the effect of histone chaperones on tumor immunity in HCC. Bioinformatics analyses were initially performed using The Cancer Genome Atlas (TCGA) database, and were validated using the Gene Expression Omnibus (GEO) database and the International Cancer Genome Consortium (ICGC) database. Immune-related histone chaperones were screened with the Spearman rank coefficient. Consensus clustering was utilized to divide the HCC samples into two clusters. ESTIMATE, CIBERSORT and ssGSEA analyses were performed to assess immune infiltration. The expression of immunomodulatory genes, chemokines and chemokine receptors was analyzed to evaluate sensitivity to immunotherapy. The differentially expressed genes (DEGs) were included in weighted gene coexpression network analysis (WGCNA) to identify the hub genes. Enrichment analyses were used to investigate the functions of the hub genes. The Kaplan-Meier method and log-rank test were conducted to draw survival curves. A Cox regression analysis was utilized to identify independent risk factors affecting prognosis. HSPA8 and DEK were screened out from 36 known histone chaperones based on their strongest correlation with the ESTIMATE score. Cluster 2, with high HSPA8 expression and low DEK expression, tended to have stronger immune infiltration and better sensitivity to immunotherapy than Cluster 1, with low HSPA8 expression and high DEK expression. Furthermore, WGCNA identified 12 hub genes closely correlated with immune infiltration from the DEGs of the two clusters, of which FBLN2 was proven to be an independent protective factor of HCC patients. HSPA8 and DEK are expected to be biomarkers for precisely predicting the effect of immunotherapy, and FBLN2 is expected to be a therapeutic target of HCC.

Bone marrow-derived mesenchymal stromal cells promote colorectal cancer cell death under low-dose irradiation.

BACKGROUND: Radiotherapy remains one of the cornerstones to improve the outcome of colorectal cancer (CRC) patients. Radiotherapy of the CRC not only help to destroy cancer cells but also remodel the tumour microenvironment by enhancing tumour-specific tropism of bone marrow-derived mesenchymal stromal cell (BM-MSC) from the peripheral circulation. However, the role of local MSCs and recruited BM-MSC under radiation were not well defined. Indeed, the functions of BM-MSC without irradiation intervention remained controversial in tumour progression: BM-MSC was previously shown to modulate the immune function of major immune cells, resulting in an impaired immunological sensitivity and to induce an increased risk of tumour recurrence. In contrast, it could also secrete various cytokines and possess anticancer effect. METHODS: Three co-cultivation modules, 3D culture modules, and cancer organoids were established. The induction of cytokines secretion in hBM-MSCs after irradiation was analysed by ELISA array and flow cytometry. AutoMac separator was used to separate hBM-MSC and CRC automatically. Cells from the co-cultured group and the control group were then irradiated by UV-C lamp and X-ray. Proliferation assay and viability assay were performed. RESULTS: In this study, we show that BM-MSCs can induce the EMT progression of CRC cells in vitro. When irradiated with low doses of ultraviolet radiation and X-rays, BM-MSCs show an anti-tumour effect by secreting certain cytokine (TNF-α, IFN-γ) that lead to the inhibition of proliferation and induction of apoptosis of CRC cells. This was further verified in a 3D culture model of a CRC cell in vitro. Furthermore, irradiation on the co-culture system induced the cleavage of caspase3, and attenuated the phosphorylation of phosphatidylinositol 3-kinase (PI3K)/AKT and extracellular signal-regulated kinase in cancer cells. The signal pathways above might contribute to the cancer cell death. CONCLUSIONS: Taken together, we show that BM-MSC can potentially promote the effect of radiotherapy in CRC.

Overexpression of CXCR2 predicts poor prognosis in patients with colorectal cancer.

Colorectal cancer is a heterogeneous disease. Although many risk factors are used to predict colorectal cancer patients' prognosis after surgical resection, new prognostic factors are still needed to be defined to promote predictive efficacy of prognosis and further guide therapies. Herein, we identified the prognostic significance of CXCR2 in colorectal cancer patients. We retrospectively analysed 134 patients with colorectal cancer who underwent minimally invasive surgery between 2010 and 2011. The overall cohort was divided into a training set (n = 78) and a validation set (n = 56). We detected CXCR2 expression using immunohistochemical staining and defined the cut-off value using X-tile program. Next, we analysed the association between CXCR2 expression and clinicopathologic features in training and validation sets. High expression of CXCR2 was associated with Dukes stage (P = 0.018), tumor invasion (P = 0.018) and liver metastasis (P = 0.047). Multivariate COX regression analyses confirmed that high CXCR2 level was an independent prognostic risk factor for both overall survival and disease free survival. Kaplan-Meier survival analysis demonstrated that patients with high expression of CXCR2 had a poor overall survival and disease free survival even in low-risk group (I + II). This indicated that CXCR2 can help to refine individual risk stratification. In addition, we established Nomograms of all significant factors to predict 3- or 5-years overall survival and disease free survival. Moreover, we found the combination of CXCR2 and its ligand CXCL5 had more significant value in predicting the prognosis than single CXCR2 factor.

Tumor-derived CXCL5 promotes human colorectal cancer metastasis through activation of the ERK/Elk-1/Snail and AKT/GSK3ß/ß-catenin pathways.

BACKGROUND: Metastasis is a major cause of death in human colorectal cancer patients. However, the contribution of chemokines in the tumor microenvironment to tumor metastasis is not fully understood. METHODS: Herein, we examinined several chemokines in colorectal cancer patients using chemokine ELISA array. Immunohistochemistry was used to detect expression of CXCL5 in colorectal cancer patients tissues. Human HCT116 and SW480 cell lines stably transfected with CXCL5, shCXCL5 and shCXCR2 lentivirus plasmids were used in our in vitro study. Immunoblot, immunofluorescence and transwell assay were used to examine the molecular biology and morphological changes in these cells. In addition, we used nude mice to detect the influence of CXCL5 on tumor metastasis in vivo. RESULTS: We found that CXCL5 was overexpressed in tumor tissues and associated with advanced tumor stage as well as poor prognosis in colorectal cancer patients. We also demonstrated that CXCL5 was primarily expressed in the tumor cell cytoplasm and cell membranes, which may indicate that the CXCL5 was predominantly produced by cancer epithelial cells instead of fibroblasts in the tumor mesenchyme. Additionally, overexpression of CXCL5 enhanced the migration and invasion of colorectal cancer cells by inducing the epithelial-mesenchymal transition (EMT) through activation of the ERK/Elk-1/Snail pathway and the AKT/GSK3ß/ß-catenin pathway in a CXCR2-dependent manner. The silencing of Snail and ß-catenin attenuated CXCL5/CXCR2-enhanced cell migration and invasion in vitro. The elevated expression of CXCL5 can also potentiate the metastasis of colorectal cancer cells to the liver in vivo in nude mice intrasplenic injection model. CONCLUSION: In conclusion, our findings support CXCL5 as a promoter of colorectal cancer metastasis and a predictor of poor clinical outcomes in colorectal cancer patients.

Cadherin-12 enhances proliferation in colorectal cancer cells and increases progression by promoting EMT.

Cadherin-12 (CDH12) is a subtype of N-cadherin family. In this study, we investigated the expression of CDH12 and the role of CDH12 in prognosis of colorectal cancer (CRC) patients. In addition, we observed the influence of CDH12 on proliferation and progression of CRC cell lines. By using immunohistochemical staining, we analyzed CRC samples and adjacent non-tumor tissues collected from 78 patients who underwent laparoscopic surgery in Shanghai Minimally Invasive Center, China. Statistical analyses were used to analyze relationship between CDH12 and tumor features. Kaplan-Meier method was used to analyze patients' survival. Proliferation ability of CRC cells was tested by CCK-8 assay, and transwell assays were performed to detect migration and invasion ability. Western blot assay was performed to investigate epithelial-mesenchymal transition (EMT) variants. We found that expression of CDH12 in tumor tissue was higher than in adjacent normal tissue. High expression of CDH12 was associated with tumor invasion depth and predicts poor prognosis of CRC patients. Ectopic/repressing expression of CDH12 increased/decreased the proliferation and migration ability of CRC cells. CDH12 is able to increase cancer cell migration and invasion via promoting EMT by targeting transcriptional factor Snail. These findings may conclude that CDH12 may act as a predictor in CRC patients' prognosis and an oncogene in CRC cell proliferation and migration. CDH12 may influence CRC cell progression through promoting EMT by targeting Snail. In addition, CDH12 is promoted by MCP1 through induction of MCPIP.

PFDN1, an indicator for colorectal cancer prognosis, enhances tumor cell proliferation and motility through cytoskeletal reorganization.

Prefoldin (PFDN) subunits have been reported upregulated in various tumor types, while the expression and functions of PFDN1 (PFDN subunit 1) in colorectal cancer (CRC) are not well elucidated. The aim of this study was to investigate the use of PFDN1 as a poor prognosis indicator for CRC and explore the functions of PFDN1 in CRC. The relationship between PFDN1 expression and CRC clinical-pathological statistics was detected on the tissue microarray containing 145 cases of CRC. ShRNA was used to silence PFDN1 expression in SW480 and RKO CRC cells, and these transfected cells were analyzed for changes in proliferation, colony formation, cell cycle, migration, and invasion. Immunofluorescence and immunoblot were used to determine the remodeling of the F-actin and α-tubulin. Finally, tumor growth on nude mice was observed and measured. In this study, we found PFDN1 was upregulated in CRC tissues compared with adjacent normal tissues. Also, PFDN1 expression positively correlated with tumor size and tumor invasion. Moreover, after silencing PFDN1 in SW480 and RKO cells, the proliferation and motility of CRC cells were significantly suppressed. The inhibitory effect of PFDN1 on tumor cell growth and motility was partially due to G2/M cell cycle blockage and cytoskeletal deficiency. Finally, in vivo assay showed that downregulation of PFDN1 inhibited tumor growth on nude mice and PFDN1 expression correlated with higher levels of Ki-67 staining. These findings indicate that PFDN1 was involved in the progression of CRC, and provide new insights into PFDN1 as a potential therapeutic target for CRC treatment.

The molecular effect of metastasis suppressors on Src signaling and tumorigenesis: new therapeutic targets.

A major problem for cancer patients is the metastasis of cancer cells from the primary tumor. This involves: (1) migration through the basement membrane; (2) dissemination via the circulatory system; and (3) invasion into a secondary site. Metastasis suppressors, by definition, inhibit metastasis at any step of the metastatic cascade. Notably, Src is a non-receptor, cytoplasmic, tyrosine kinase, which becomes aberrantly activated in many cancer-types following stimulation of plasma membrane receptors (e.g., receptor tyrosine kinases and integrins). There is evidence of a prominent role of Src in tumor progression-related events such as the epithelial-mesenchymal transition (EMT) and the development of metastasis. However, the precise molecular interactions of Src with metastasis suppressors remain unclear. Herein, we review known metastasis suppressors and summarize recent advances in understanding the mechanisms of how these proteins inhibit metastasis through modulation of Src. Particular emphasis is bestowed on the potent metastasis suppressor, N-myc downstream regulated gene 1 (NDRG1) and its interactions with the Src signaling cascade. Recent studies demonstrated a novel mechanism through which NDRG1 plays a significant role in regulating cancer cell migration by inhibiting Src activity. Moreover, we discuss the rationale for targeting metastasis suppressor genes as a sound therapeutic modality, and we review several examples from the literature where such strategies show promise. Collectively, this review summarizes the essential interactions of metastasis suppressors with Src and their effects on progression of cancer metastasis. Moreover, interesting unresolved issues regarding these proteins as well as their potential as therapeutic targets are also discussed.

Challenge or opportunity: outcomes of laparoscopic resection for rectal cancer in patients with high operative risk.

This study investigated the impact of laparoscopic rectal cancer resection for patients with high operative risk, which was defined as American Society of Anesthesiology (ASA) grades III and IV. This study was conducted at a single center on patients undergoing rectal resection from 2006 to 2010. After screening by ASA grade III or IV, 248 patients who met the inclusion criteria were identified, involving 104 open and 144 laparoscopic rectal resections. The distribution of the Charlson Comorbidity Index was similar between the two groups. Compared with open rectal resection, laparoscopic resection had a significantly lower total complication rate (P<.0001), lower pain rate (P=.0002), and lower blood loss (P<.0001). It is notable that the two groups of patients had no significant difference in cardiac and pulmonary complication rates. Thus, these data showed that the laparoscopic group for rectal cancer could provide short-term outcomes similar to those of their open resection counterparts with high operative risk. The 5-year actuarial survival rates were 0.8361 and 0.8119 in the laparoscopic and open groups for stage I/II (difference not significant), as was the 5-year overall survival rate in stage III/IV (P=.0548). In patients with preoperative cardiovascular or pulmonary disease, the 5-year survival curves were significantly different (P=.0165 and P=.0210), respectively. The cost per patient did not differ between the two procedures. The results of this analysis demonstrate the potential advantages of laparoscopic rectal cancer resection for high-risk patients, although a randomized controlled trial should be conducted to confirm the findings of the present study.

Antitumor efficacy of CC motif chemokine ligand 19 in colorectal cancer.

OBJECTIVES: To investigate the function of CC motif chemokine ligand 19 (CCL19) in colorectal cancer (CRC). METHODS: Quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blot and immunohistochemistry were performed separately to detect the expression of CCL19 in colorectal carcinoma tissues. The expression of CCL19 and its receptor (CCR7) in CRC cell lines were screened by Western blot. SW620, SW1116 and LoVo cell lines were screened and processed with recombinant human CCL19 (rhCCL19) or si-CCL19 RNA. Cell proliferation assay and transwell assay were performed to evaluate the proliferation, migration and invasion of CRC cells, respectively. And the role of proangiogenesis was checked by endothelial tube formation assay. RESULTS: qRT-PCR, Western blot and immunohistochemistry revealed that both CCL19 mRNA and protein were obviously expressed in a lower degree in CRC tissues than normal tissues (P < 0.01). The CCL19 expression correlated with tumor size (P = 0.03) and invasion depth (P = 0.04) in a negative manner and CCL19-positive patients had longer lifespans (P < 0.05). SW620 and SW1116 cells were screened as CCL19/CCR7 high-expression cells, while LoVo was selected as CCL19/CCR7 low-expression cell among seven CRC cell lines by Western blot. The proliferation, migration, invasion and proangiogenesis of SW620 and SW1116 cells were distinctly suppressed after they were stimulated by rhCCL19 (P < 0.05), and the data presented dose-dependency. Oppositely, these abilities were significantly enhanced after CCL19 gene was silenced (P < 0.05). However, the effects of rhCCL19 and si-CCL19 RNA on LoVo were not significant (P > 0.05). CONCLUSION: Our research findings indicate that CCL19 may play a suppressive role in colorectal tumorigenesis.

Long-term outcome of laparoscopic-assisted right-hemicolectomy with D3 lymphadenectomy versus open surgery for colon carcinoma.

PURPOSE: To investigate the applicability, safety, short-term and long-term outcomes of laparoscopic surgery in the treatment of right-sided colon carcinomas with D3 lymphadenectomy. METHODS: Between June 2003 and September 2010, 324 patients with right-sided colon carcinoma underwent surgical treatment in the same hospital, 177 cases were treated by laparoscopic surgery (LRH group) and 147 cases by open surgery (ORH group). We performed a retrospective analysis of the differences between the two groups in terms of the clinical data. RESULTS: There were no significant differences between the two groups in the demographic data; however, the recovery time was significantly shorter in the LRH group, the number of overall lymph nodes harvested and principle lymph nodes harvested in the LRH group was significantly higher than in the ORH group, the incidence of postoperative complications was 12.99 % in the LRH group and 22.45 % in the ORH group (P < 0.05), and the recurrence rate in the LRH group was lower than that in the ORH group, although the difference was not significant (15.25 vs 19.73 %). The cumulative overall survival for all stages at 1, 3 and 5 years in the LRH group (97.18, 83.73 and 70.37 %) were not significantly different compared to those in the ORH group (94.56, 77.84 and 66.97 %). CONCLUSIONS: Laparoscopic-assisted right hemicolectomy with D3 lymphadenectomy for colon carcinomas is safe and effective, while it is also superior to open surgery regarding the short-term outcomes, and the long-term outcomes are similar to those of open surgery.

Cadherin-12 contributes to tumorigenicity in colorectal cancer by promoting migration, invasion, adhersion and angiogenesis.

BACKGROUND: Cadherin 12 (CDH12), which encodes a type II classical cadherin from the cadherin superfamily, may mediate calcium-dependent cell adhesion. It has been demonstrated that CDH12 could play an important role in the invasion and metastasis of salivary adenoid cystic carcinoma. We decided to investigate the relationship between CDH12 expression level and clinicopathologic variables in colorectal carcinoma (CRC) patients and to explore the functions of CDH12 in tumorigenesis in CRC. METHODS: The expression levels of CDH12 in colorectal carcinoma tissues were detected by immunohistochemistry. Real-time PCR and Western Blot were used to screen CDH12 high-expression cell lines. CCK-8 assay was used to detect the proliferation ability of CRC cells being transfected by shRNAs against CDH12. The wound assay and transwell assay were performed to test migration and invasion ability. The importance of CDH12 in cell-cell junctions was detected by cell adhesion assay and cell aggregation assay. Endothelial tube formation assay was used to test the influence of CDH12 on angiogenesis. RESULTS: Statistical analysis of clinical cases revealed that the positive rate of CDH12 was higher in the CRC tumor tissues compared with the adjacent non-tumor tissues. The expression levels of CDH12 in CRC patients are significantly correlated with invasion depth. Consistently, the ability of proliferation, migration and invasion were suppressed when CDH12 was decreased in CRC cells transfected with shRNAs. Cell adhesion assay and cell aggregation assay presented that tumor cells tend to disperse with the lack of CDH12. Endothelial tube formation assay showed that down-regulation of CDH12 could obviously inhibit the process of angiogenesis, implying that CDH12 may play an important role in tumor metastasis CONCLUSION: Our results showed that CDH12 promotes proliferation, migration, invasion, adhesion and angiogenesis, suggesting that CDH12 may be an oncogene in colorectal cancer. CDH12 is expected to become a new diagnostic and prognostic marker and a novel target of the treatment of colorectal cancer.

Long-term results of laparoscopy-assisted radical right hemicolectomy with D3 lymphadenectomy: clinical analysis with 177 cases.

PURPOSES: To study the feasibility, safety, and short-/long-term outcomes of laparoscopy-assisted right hemicolectomy with D3 lymphadenectomy for colon cancer. METHODS: The clinical data of 177 cases that underwent laparoscopy-assisted radical right hemicolectomy with D3 lymphadenectomy for colon cancer between Jun 2003 and Sep 2010 was collected; the safety of operation, status of recovery, complication, oncological outcomes, and results of short-/long-term follow-up were analyzed. RESULTS: No case died in this study; five cases (2.82 %) were converted to open surgery. Four cases (2.26 %) underwent hand-assisted laparoscopic right hemicolectomy. The average operation time was 133 ± 36 min, and the blood loss was 94 ± 34 ml. The average time for passage of flatus, liquid food eating, and hospitalization were 2.1 ± 0.7, 3.2 ± 0.5, and 10.4 ± 2.7 day, respectively. The total number of lymph nodes removed was 15.2 ± 10.1. Postoperative complications were observed in 23 of 177 patients (12.99 %). The median follow-up period was 54 months; port-site recurrence was observed in one patient; local recurrence was found in five cases (2.82 %); distant metastasis was found in 21 cases (11.86 %). The cumulative overall survival of all stages at 12, 36, 60, and 72 months was 97.18 %, 83.73 %, 70.37 %, and 68.99 %, respectively. The cancer-specific survival was 98.73 % (12 months), 87.81 % (36 months), and 80.17 % (60 months). CONCLUSIONS: Laparoscopy-assisted right hemicolectomy with D3 lymphadenectomy can be successfully performed for right colon cancer with the advantages of minimally invasive surgery. Moreover, the results implied appropriate short- and long-term outcomes.

Emerging roles of the ribonucleotide reductase M2 in colorectal cancer and ultraviolet-induced DNA damage repair.

AIM: To investigate the roles of the ribonucleotide reductase M2 (RRM2) subunit in colorectal cancer (CRC) and ultraviolet (UV)-induced DNA damage repair. METHODS: Immunohistochemical staining of tissue microarray was performed to detect the expression of RRM2. Seven CRC cell lines were cultured and three human colon cancer cell lines, i.e., HCT116, SW480 and SW620, were used. Reverse transcription polymerase chain reaction and Western blotting were performed to determine the mRNA and protein expression levels of RRM2, respectively. Cell proliferation assay, cell cycle analysis were performed. Cell apoptosis was evaluated by double staining with fluorescein isothiocyanate-conjugated Annexin V and propidium iodide (PI) using Annexin V/PI apoptosis kit. The motility and invasion of CRC cells were assessed by the Transwell chamber assay. Cells were irradiated with a 254 nm UV-C lamp to detect the UV sensitivity after RRM2 depletion. RESULTS: Immunohistochemical staining revealed elevated RRM2 levels in CRC tissues. RRM2 overexpression was positively correlated with invasion depth (P < 0.05), poorly differentiated type (P = 0.0051), and tumor node metastasis stage (P = 0.0015). The expression of RRM2 in HCT116 cells was downregulated after transfection, and HCT116 cell proliferation was obviously suppressed compared to control groups (P < 0.05). In the invasion test, the number of cells that passed through the chambers in the RRM2-siRNA group was 81 ± 3, which was lower than that in the negative control (289 ± 7) and blank control groups (301 ± 7.2). These differences were statistically significant (P < 0.01). Our data suggest that RRM2 overexpression may be associated with CRC progression. RRM2 silencing by siRNA may inhibit the hyperplasia and invasiveness of CRC cells, suggesting that RRM2 may play an important role in the infiltration and metastasis of CRC, which is a potential therapeutic strategy in CRC. In addition, RRM2 depletion increased UV sensitivity. CONCLUSION: These findings suggest that RRM2 may be a facilitating factor in colorectal tumorigenesis and UV-induced DNA damage repair.

Polo-like kinase 1 is overexpressed in colorectal cancer and participates in the migration and invasion of colorectal cancer cells.

BACKGROUND: Polo-like kinase 1 (PLK1) is an important molecule in proliferation of many human cancers. The aim of study is to clarify the expression patterns and potential function of PLK1 in colorectal cancers. MATERIAL/METHODS: Fifty-six colorectal cancers samples were collected and arranged onto a tissue array and the expression of PLK1 were detected by immunohistochemistry and correlated with clinico-pathological characteristics and expression of PCNA. Expression of PLK1 in 9 colorectal cancer cells lines was investigated by RT-PCR and Western blot, then SW1116 cells lines were treated with PLK1 siRNA and the efficiency was examined by Western blot. Transwell test was applied to detect the migration and invasion capability of cancer cells by counting the number of cells passing through the membranes. Cell proliferation and apoptosis were examined by Cell Counting Kit-8 (CCK-8) and Annexin-V Kit. RESULTS: PLK1 was positively expressed in 73.2% (41/56) of colorectal cancers tissues, but in only 3.6% (2/56) of normal tissues, and was associated with Duke's stage (P<0.01), tumor size (P<0.01), invasion extent (P<0.05) and lymphatic metastasis (P<0.01). The expression of PLK1 was correlated with expression of PCNA (R=0.553, P<0.01). PLK1 was inhibited in SW1116 cells by treating with PLK1 siRNA oligos, which resulted in a decreased number of cells passing through the membrane as compared with control groups (P<0.01) at 24 hours after transfection. Cell proliferation was inhibited from 48 hours after transfection, while cells apoptosis was induced from 72 hours after transfection. CONCLUSIONS: PLK1 could be a progression marker for colorectal cancer patients and PLK1 depletion can inhibit migration and invasion capability of colorectal cancer cells SW1116, suggesting that PLK1 might be involved in metastasis and invasion of colorectal cancer. Therapeutic strategies targeting PLK1 may be a new approach to colorectal cancer.