Low skeletal muscle radiodensity is a risk factor for adjuvant chemotherapy discontinuation in colorectal cancer.

BACKGROUND: Previously, we reported SMR (skeletal muscle radiodensity) as a potential prognostic marker for colorectal cancer. However, there have been limited studies on the association between SMR and the continuation of adjuvant chemotherapy in colorectal cancer. METHODS: In this retrospective study, 143 colorectal cancer patients underwent curative surgery and adjuvant chemotherapy using the CAPOX regimen. Patients' SMRs were measured from preoperative CT images and divided into low (bottom quarter) and high (top three quarters) SMR groups. We compared chemotherapy cycles, capecitabine and oxaliplatin doses, and adverse effects in each group. RESULTS: The low SMR group had significantly fewer patients completing adjuvant chemotherapy compared to the high SMR group (44% vs. 68%, P < 0.01). Capecitabine and oxaliplatin doses were also lower in the low SMR group. Incidences of Grade 2 or Grade 3 adverse effects did not differ between groups, but treatment discontinuation due to adverse effects was significantly higher in the low SMR group. Logistic regression analysis revealed Stage III disease (odds ratio 18.09, 95% CI 1.41-231.55) and low SMR (odds ratio 3.26, 95% CI 1.11-9.56) as factors associated with unsuccessful treatment completion. Additionally, a higher proportion of low SMR patients received fewer than 2 cycles of chemotherapy (50% vs. 12%). CONCLUSION: The low SMR group showed higher treatment incompletion rates and received lower drug doses during adjuvant chemotherapy. Low SMR independently contributed to treatment non-completion in colorectal cancer patients.

Less demand on stem cell marker-positive cancer cells may characterize metastasis of colon cancer.

BACKGROUND: CD44 and CD133 are stem cell markers in colorectal cancer (CRC). CD44 has distinctive isoforms with different oncological properties like total CD44 (CD44T) and variant CD44 (CD44V). Clinical significance of such markers remains elusive. METHODS: Sixty colon cancer were examined for CD44T/CD44V and CD133 at mRNA level in a quantitative PCR, and clarified for their association with clinicopathological factors. RESULTS: (1) Both CD44T and CD44V showed higher expression in primary colon tumors than in non-cancerous mucosas (p<0.0001), while CD133 was expressed even in non-cancerous mucosa and rather decreased in the tumors (p = 0.048). (2) CD44V expression was significantly associated with CD44T expression (R = 0.62, p<0.0001), while they were not correlated to CD133 at all in the primary tumors. (3) CD44V/CD44T expressions were significantly higher in right colon cancer than in left colon cancer (p = 0.035/p = 0.012, respectively), while CD133 expression were not (p = 0.20). (4) In primary tumors, unexpectedly, CD44V/CD44T/CD133 mRNA expressions were not correlated with aggressive phenotypes, but CD44V/CD44T rather significantly with less aggressive lymph node metastasis/distant metastasis (p = 0.040/p = 0.039, respectively). Moreover, both CD44V and CD133 expressions were significantly decreased in liver metastasis as compared to primary tumors (p = 0.0005 and p = 0.0006, respectively). CONCLUSION: Our transcript expression analysis of cancer stem cell markers did not conclude that their expression could represent aggressive phenotypes of primary and metastatic tumors, and rather represented less demand on stem cell marker-positive cancer cells.

Short-term outcomes of robot-assisted versus conventional laparoscopic surgery for mid and low rectal cancer after neoadjuvant chemoradiotherapy: a propensity score-matched analysis.

The benefits of robot-assisted laparoscopic surgery (RALS) for rectal cancer remain controversial. Only a few studies have evaluated the safety and feasibility of RALS following neoadjuvant chemoradiotherapy (NCRT). This study aimed to compare the short-term outcomes of RALS versus conventional laparoscopic surgery (CLS) after NCRT for rectal cancer. Propensity score matching of 111 consecutive patients who underwent RALS or CLS after NCRT for rectal adenocarcinoma between February 2014 and February 2022 was performed. Among them, 60 matched patients were enrolled and their short-term outcomes were compared. Although operative time, conversion rate to open laparotomy and blood loss were comparable, the incidence of postoperative complications, including anastomotic leakage, was significantly lower, urinary retention tended to be lower, and the days to soft diet intake and postoperative hospital stay were significantly shorter in the RALS than the CLS group. No postoperative mortality was observed in either group, and there were no significant differences in terms of resection margins and number of lymph nodes dissected. RALS after NCRT for rectal cancer is safe and technically feasible, and has acceptable short-term outcomes. Further studies are required for validation of the long-term oncological outcomes.

Short- and long-term outcomes of robotic-assisted laparoscopic surgery for rectal cancer: A single-center retrospective cohort study.

INTRODUCTION: Whether rectal cancer surgery by robotic-assisted laparoscopic surgery provides beneficial advantages remains controversial. Although favorable outcomes in terms of the safety and technical feasibility of robotic-assisted laparoscopic surgery have been demonstrated for rectal cancer, long-term oncological outcomes for robotic-assisted laparoscopic surgery have only been examined in a few studies. This retrospective study of subjects who underwent robotic-assisted laparoscopic surgery evaluated short- and long-term outcomes of consecutive rectal cancer patients. METHODS: Between November 2016 and January 2020, we analyzed the records of 62 consecutive patients who underwent robotic-assisted laparoscopic surgery for rectal adenocarcinoma without distant metastasis to evaluate short- and long-term outcomes. RESULTS: Tumors were located in the lower or mid-rectum (88.7%) in most patients. The median operative time was 357 min. No patient received transfusions, and the median blood loss was 10.5 ml. Open laparotomy was not required in any patient. A Clavien-Dindo classification of all grades was observed in 12 patients (19.4%). Positive radial margin was not observed in any patient. Duration of median follow-up was 40.5 mo, while 3-y overall survival and 3-y relapse-free survival rates were 96.8% and 85.0%, respectively. The local recurrence rate was 3.4%. CONCLUSION: Favorable short- and long-term outcomes demonstrated robotic-assisted laparoscopic surgery was safe and technically feasible for rectal cancer.

Skeletal Muscle Changes Assessed by Preoperative Computed Tomography Images Can Predict the Long-Term Prognosis of Stage III Colorectal Cancer.

Background: Myopenia and myosteatosis are reported to be long-term prognostic factors in patients with colorectal cancer (CRC). However, the established parameters are unsuitable for the Japanese population because their body composition is different from that of the Western population. Objective: We aimed to elucidate the effect of skeletal muscle changes among Japanese adults, measured using preoperative computed tomography (CT) as a prognostic factor in patients with stage III CRC. Patients: We retrospectively analyzed 341 patients diagnosed with stage III CRC. The cross-sectional area (skeletal muscle index: SMI) and mean radiodensity of skeletal muscle (skeletal muscle radiodensity: SMR) were measured using preoperative CT. The optimal sex-specific cutoff value, which was used to divide the patients according to the risk of recurrence, was set for SMI and SMR. Univariate and multivariate analysis were performed to determine the prognostic factors for recurrence-free survival (RFS). Results: The cutoff values of SMI for men and women were set as 48.5 and 41.4, respectively, and those of SMR were 35.0 and 21.7, respectively. Univariate analysis identified low SMI and SMR in men and low SMR in women as the worst prognostic factors for RFS. Multivariate analysis identified low SMI in men and low SMR in women as independent poor prognostic factors for RFS (hazard ratio [HR] = 1.87, 95% confidence interval [CI], 1.08-3.47, P = .03 and HR = 2.49, CI, 1.21-4.95, P = .01). Conclusion: Low SMI in men and low SMR in women were the independent prognostic factors for patients with stage III CRC.

Comparison of short-term outcomes of robotic-assisted and conventional laparoscopic surgery for rectal cancer: A propensity score-matched analysis.

INTRODUCTION: The advantages of robotic-assisted laparoscopic surgery (RALS) for rectal cancer remain controversial. This study clarified and compared the short-term outcomes of RALS for rectal cancer with those of conventional laparoscopic surgery (CLS). METHODS: The records of 303 consecutive patients who underwent RALS or CLS for rectal adenocarcinoma between November 2016 and November 2021 were analyzed using propensity score-matched analysis. After matching, 188 patients were enrolled in our study to compare short-term outcomes, such as operative results, postoperative complications, and pathological findings, in each group. RESULTS: After matching, baseline characteristics were comparable between groups. Although operative time in the RALS group was significantly longer than in the CLS group (p < 0.0001), the conversion rate to open laparotomy and the postoperative complication rate in the RALS group were significantly lower than in the CLS group (p = 0.0240 and p = 0.0109, respectively). Blood loss was comparable between groups. In the RALS group, postoperative hospital stay and days to soft diet were significantly shorter than those in the CLS group (p = 0.0464 and p < 0.0001, respectively). No postoperative mortality was observed in either group and significant differences were observed in resection margins and number of lymph nodes harvested. CONCLUSION: Robotic-assisted laparoscopic surgery for rectal cancer was safe, technically feasible, and had acceptable short-term outcomes. Further studies are required to validate long-term oncological outcomes.

EP3 signaling in dendritic cells promotes liver repair by inducing IL-13-mediated macrophage differentiation in mice.

Macrophage plasticity is essential for liver wound healing; however, the mechanisms underlying macrophage phenotype switching are largely unknown. Dendritic cells (DCs) are critical initiators of innate immune responses; as such, they orchestrate inflammation following hepatic injury. Here, we subjected EP3-deficient (Ptger3-/- ) and wild-type (WT) mice to hepatic ischemia-reperfusion (I/R) and demonstrate that signaling via the prostaglandin E (PGE) receptor EP3 in DCs regulates macrophage plasticity during liver repair. Compared with WT mice, Ptger3-/- mice showed delayed liver repair accompanied by reduced expression of hepatic growth factors and accumulation of Ly6Clow reparative macrophages and monocyte-derived DCs (moDCs). MoDCs were recruited to the boundary between damaged and undamaged liver tissue in an EP3-dependent manner. Adoptive transfer of moDCs from Ptger3-/- mice resulted in impaired repair, along with increased numbers of Ly6Chigh inflammatory macrophages. Bone marrow macrophages (BMMs) up-regulated expression of genes related to a reparative macrophage phenotype when co-cultured with moDCs; this phenomenon was dependent on EP3 signaling. In the presence of an EP3 agonist, interleukin (IL)-13 derived from moDCs drove BMMs to increase expression of genes characteristic of a reparative macrophage phenotype. The results suggest that EP3 signaling in moDCs facilitates liver repair by inducing IL-13-mediated switching of macrophage phenotype from pro-inflammatory to pro-reparative.

Lymphangiogenesis and accumulation of reparative macrophages contribute to liver repair after hepatic ischemia-reperfusion injury.

Hepatic tissue repair plays a critical role in determining the outcome of hepatic ischemia-reperfusion (I/R) injury. Hepatic lymphatics participate in the clearance of dead tissues and contribute to the reparative process after acute hepatic injury; however, it remains unknown whether lymphangiogenesis in response to hepatic inflammation is involved in liver repair. Herein, we determined if hepatic lymphangiogenesis improves liver repair after hepatic I/R injury. Using a mouse model of hepatic I/R injury, we investigated hepatic lymphatic structure, growth, and function in injured murine livers. Hepatic I/R injury enhanced lymphangiogenesis around the portal tract and this was associated with increased expression of pro-lymphangiogenic growth factors including vascular endothelial growth factor (VEGF)-C and VEGF-D. Recombinant VEGF-D treatment facilitated liver repair in association with the expansion of lymphatic vessels and increased expression of genes related to the reparative macrophage phenotype. Treatment with a VEGF receptor 3 (VEGFR3) inhibitor suppressed liver repair, lymphangiogenesis, drainage function, and accumulation of VEGFR3-expressing reparative macrophages. VEGF-C and VEGF-D upregulated expression of genes related to lymphangiogenic factors and the reparative macrophage phenotype in cultured macrophages. These results suggest that activation of VEGFR3 signaling increases lymphangiogenesis and the number of reparative macrophages, both of which play roles in liver repair. Expanded lymphatics and induction of reparative macrophage accumulation may be therapeutic targets to enhance liver repair after hepatic injury.

WiNTRLINC1/ASCL2/c-Myc Axis Characteristics of Colon Cancer with Differentiated Histology at Young Onset and Essential for Cell Viability.

BACKGROUND: WiNTRLINC1 is a long non-coding RNA (lncRNA) that positively regulates the Wnt pathway via achaete-scute complex homolog 2 (ASCL2) in colorectal cancer. ASCL2 was recently reported to play a critical role in chemoresistance, however clinical relevance of the WiNTRLINC1/ASCL2 axis remains obscure in colon cancer. PATIENTS AND METHODS: WiNTRLINC1/ASCL2 expression was investigated at messenger RNA (mRNA) level in 40 primary colon cancer tissues and the corresponding normal mucosa tissues, together with Wnt-related genes (c-Myc/PRL-3) and other lncRNAs (H19, HOTAIR, and MALAT1). Knock-down experiments of WiNTRLINC1 clarified its role in their expression and chemoresistance. RESULTS: Real-time quantitative reverse transcriptase-polymerase chain reaction confirmed definite overexpression of WiNTRLINC1 mRNA in primary colon cancer compared with the corresponding normal colon mucosa tissues (p = 0.0005), such as ASCL2, c-Myc, and PRL-3 (p < 0.0001). The four gene expression signatures were tightly associated in the center of the ASCL2 gene (r = 0.72, p < 0.0001) in clinical samples. WiNTRLINC1 was not significantly associated with prognostic factors in colon cancer and other lncRNAs, while the WiNTRLINC1/ASCL2/c-Myc signatures were unique to young-onset colon cancer with differentiated histology. On the other hand, undifferentiated histology was significantly associated with H19 expression. Knockdown of the WiNTRLINC1 gene reduced the expression of ASCL2/c-Myc, but rather augmented PRL-3 at mRNA level, and robustly affected cell viability in colon cancer cell lines. CONCLUSION: The enhanced WiNTRLINC1/ASCL2/c-Myc axis involved in Wnt pathway activation is a common pathway essential for differentiated colon tumorigenesis, especially with young onset, and may be essential for a viable phenotype of colon cancer.

Thromboxane A2 receptor signaling in endothelial cells attenuates monocrotaline-induced liver injury.

Sinusoidal obstruction syndrome (SOS) is a major complication of chemotherapy and hematopoietic stem cell transplantation. The early stage of SOS is characterized by liver sinusoidal endothelial cell (LSEC) injury accompanied by platelet aggregation. Thromboxane A2 (TxA2) induces platelet aggregation through the thromboxane prostanoid (TP) receptor. In this study, we explored the role of TP signaling in a monocrotaline (MCT)-induced mouse model of SOS. Relative to wild-type (WT) mice, TP-deficient (TP-/-) mice exhibited more severe MCT-liver injury, as indicated by elevated levels of alanine aminotransferase (ALT) and coagulative necrosis. Extensive accumulation of platelets in the liver was observed in both WT and TP-/- mice. TP expression co-localized with CD31-positive LSECs. MCT treatment caused LSEC destruction, concomitant with elevated expression of matrix metalloproteinases (MMPs) and adhesion molecules in WT mice, and LSEC damage was further exacerbated in TP-/- mice. Viability of isolated LSECs was lower in cells from TP-/- mice, whereas mRNA levels of MMPs and adhesion molecules were higher; U46619, a TxA2 agonist, reduced these levels in WT mice. These data suggest that TP signaling has no effect on platelet accumulation during MCT-induced liver injury, but instead prevents injury by suppressing LSEC damage.

Comprehensive Genetic Search to Clarify the Molecular Mechanism of Drug Resistance Identifies ASCL2-LEF1/TSPAN8 Axis in Colorectal Cancer.

BACKGROUND: Treatment-resistance genes limiting anticancer therapy have not been well clarified in colorectal cancer (CRC). We explored gene expression profiles to identify biomarkers for predicting treatment resistance to an anticancer drug in CRC. METHODS: Six CRC cell lines were treated with phenylbutyrate (PB). The gene expression profiles were then compared using microarrays (harboring 54,675 genes), and genes associated with PB resistance were identified. Candidate genes were functionally examined in cell lines and clinically validated for treatment resistance in clinical samples. RESULTS: Both DLD1 and HCT15 cells were PB resistant, while HCT116 cells were identified as PB sensitive. On microarray analysis, among the PB resistance-related genes, the expression of the genes ASCL2, LEF1, and TSPAN8 was clearly associated with PB resistance. PB-sensitive cells transfected with one of these three genes exhibited significant (P < 0.001) augmentation of PB resistance; ASCL2 induced expression of both LEF1 and TSPAN8, while neither LEF1 nor TSPAN8 induced ASCL2. RNA interference via ASCL2 knockdown made PB-resistant cells sensitive to PB and inhibited both genes. ASCL2 knockdown also played a critical role in sensitivity to treatment by 5-fluorouracil and radiotherapy in addition to PB. Finally, ASCL2 expression was significantly correlated with histological grade of rectal cancer with preoperative chemoradiation therapy. CONCLUSIONS: ASCL2 was identified as a causative gene involved in therapeutic resistance against anticancer treatments in CRC.

Epigenetic Status of CDO1 Gene May Reflect Chemosensitivity in Colon Cancer with Postoperative Adjuvant Chemotherapy.

BACKGROUND: Cysteine dioxygenase type 1 (CDO1) acts as a tumor suppressor gene, and its expression is regulated by promoter DNA methylation in human cancer. The metabolic product mediated by CDO1 enzyme increases mitochondrial membrane potential (MMP), putatively representing chemoresistance. The aim of this study is to investigate the functional relevance of CDO1 gene in colon cancer with chemotherapy. PATIENTS AND METHODS: We investigated 170 stage III colon cancer patients for CDO1 methylation by using quantitative methylation-specific polymerase chain reaction (PCR). To elucidate the functional role of CDO1 gene in colorectal cancer (CRC) biology, we established cell lines that stably express CDO1 gene and evaluated chemosensitivity, MMP, and tolerability assay including anaerobic environment. RESULTS: Hypermethylation of CDO1 gene was an independent prognostic factor for stage III colon cancer on multivariate prognostic analysis. Surprisingly, patients with CDO1 hypermethylation exhibited better prognosis than those with CDO1 hypomethylation in stage III colon cancer with postoperative chemotherapy (P = 0.03); however, a similar finding was not seen in those without postoperative chemotherapy. In some CRC cell lines, forced expression of CDO1 gene increased MMP accompanied by chemoresistance and/or tolerance under hypoxia. CONCLUSION: CDO1 methylation may be a useful biomarker to increase the number of stage III colon cancer patients who can be saved by adjuvant therapy. Such clinical relevance may represent the functionally oncogenic property of CDO1 gene through MMP activity.

Inhibition of microsomal prostaglandin E synthase-1 facilitates liver repair after hepatic injury in mice.

BACKGROUND & AIMS: Liver repair following hepatic ischemia/reperfusion (I/R) injury is crucial to survival. This study aims to examine the role of endogenous prostaglandin E2 (PGE2) produced by inducible microsomal PGE synthase-1 (mPGES-1), a terminal enzyme of PGE2 generation, in liver injury and repair following hepatic I/R. METHODS: mPGES-1 deficient (Ptges-/-) mice or their wild-type (WT) counterparts were subjected to partial hepatic ischemia followed by reperfusion. The role of E prostanoid receptor 4 (EP4) was then studied using a genetic knockout model and a selective antagonist. RESULTS: Compared with WT mice, Ptges-/- mice exhibited reductions in alanine aminotransferase (ALT), necrotic area, neutrophil infiltration, chemokines, and proinflammatory cytokine levels. Ptges-/- mice also showed promoted liver repair and increased Ly6Clow macrophages (Ly6Clow/CD11bhigh/F4/80high-cells) with expression of anti-inflammatory and reparative genes, while WT mice exhibited delayed liver repair and increased Ly6Chigh macrophages (Ly6Chigh/CD11bhigh/F4/80low-cells) with expression of proinflammatory genes. Bone marrow (BM)-derived mPGES-1-deficient macrophages facilitated liver repair with increases in Ly6Clow macrophages. In vitro, mPGES-1 was expressed in macrophages polarized toward the proinflammatory profile. Mice treated with the mPGES-1 inhibitor Compound III displayed increased liver protection and repair. Hepatic I/R enhanced the hepatic expression of PGE receptor subtype, EP4, in WT mice, which was reduced in Ptges-/- mice. A selective EP4 antagonist and genetic deletion of Ptger4, which codes for EP4, accelerated liver repair. The proinflammatory gene expression was upregulated by stimulation of EP4 agonist in WT macrophages but not in EP4-deficient macrophages. CONCLUSIONS: These results indicate that mPGES-1 regulates macrophage polarization as well as liver protection and repair through EP4 signaling during hepatic I/R. Inhibition of mPGES-1 could have therapeutic potential by promoting liver repair after acute liver injury. LAY SUMMARY: Hepatic ischemia/reperfusion injury is a serious complication that occurs in liver surgery. Herein, we demonstrated that inducible prostaglandin E2 synthase (mPGES-1), an enzyme involved in synthesizing prostaglandin E2, worsens the injury and delays liver repair through accumulation of proinflammatory macrophages. Inhibition of mPGES-1 offers a potential therapy for both liver protection and repair in hepatic ischemia/reperfusion injury.

DNA diagnosis of peritoneal fluid cytology test by CDO1 promoter DNA hypermethylation in gastric cancer.

BACKGROUND: Minimal residual disease of the peritoneum is challenging for early cancer detection in gastric cancer (GC). Utility of PCR amplification of cancer-derived DNA has been considered feasible due to its molecular stability, however such markers have never been available in GC clinics. We recently discovered cancer-specific methylation of CDO1 gene in GC, and investigated the clinical potential to detect the minimal residual disease. METHODS: One hundred and two GC patients were investigated for peritoneal fluid cytology test (CY), and detection level of the promoter DNA methylation of CDO1 gene was assessed by quantitative methylation specific PCR (Q-MSP) in the sediments (DNA CY). RESULTS: (1) CY1 was pathologically confirmed in 8 cases, while DNA CY1 was detected in 18 cases. All 8 CY1 were DNA CY1. (2) DNA CY1 was recognized in 14.3, 25.0, 20.0, and 42.9%, in macroscopic Type II, small type III, large type III, and type IV, respectively, while it was not recognized in Type 0/I/V. (3) DNA CY1 was prognostic relevance in gastric cancer (p = 0.0004), and its significance was robust among Type III/IV gastric cancer (p = 0.006 for overall survival and p = 0.0006 for peritoneal recurrence free survival). (4) The peritoneal recurrence was hardly seen in GC patients with potent perioperative chemotherapy among those with DNA CY1. CONCLUSIONS: DNA CY1 detected by Q-MSP for CDO1 gene promoter DNA methylation has a great potential to detect minimal residual disease of the peritoneum in GC clinics as a novel DNA marker.

Lymphatic Permeation Predicts Systemic Recurrence in Combination with Vascular Involvement in Laparoscopically Resected N0 Colon Cancer.

High-risk patient selection is required in N0 colon cancer. Although a number of studies have suggested high-risk clinicopathological predictors, most of these are based on analyses in heterogeous patients in terms of surgical procedures. Laparoscopic surgery for colon cancer is becoming a standard procedure worldwide because of its less invasiveness. Accordingly, we aimed to identify bona fide high-risk factors of recurrence in homogeneous N0 patients who underwent laparoscopic surgery. Two hundred and twenty-five patients who underwent laparoscopic curative resection for N0 colon cancer were analyzed. Clinicopathological parameters were tested for their relation to survival. The 5-year recurrence-free survival rate (RFS) was 96.1 per cent. Lymphatic involvement (P < 0.001), vascular involvement (P = 0.007), and size of tumor (P = 0.023) were significantly associated with worse prognosis in the univariate analyses. Lymphatic involvement was the independent prognostic factor associated with RFS in the multivariate analysis (P = 0.013). Importantly, lymphatic involvement predicts detrimental prognosis only when vascular involvement is present. The RFS of the patients with both lymphatic and vascular involvement was 88.9 per cent, whereas it was 100 per cent in the counterpart. Differentiation, vascular involvement preoperative carcinoembryonic antigen, and CA 19-9 levels were significantly associated with lymphatic involvement in a multivariate logistic regression analyses. The present study concludes that lymphatic involvement in the presence of vascular involvement may be a high risk for systemic recurrence in the laparoscopically resected N0 colon cancer.

Promoter DNA methylation of CDO1 gene and its clinical significance in esophageal squamous cell carcinoma.

We have demonstrated that CDO1 methylation is frequently found in various cancers, including esophageal squamous cell carcinoma (ESCC), but its clinical relevance has remained elusive. CDO1 methylation was investigated in 169 ESCC patients who underwent esophagectomy between 1996 and 2007. CDO1 methylation was assessed by Q-MSP (quantitative methylation specific PCR), and its clinical significance, including its relationship to prognosis, was analyzed. (i) The median TaqMeth value of CDO1 methylation was 9.4, ranging from 0 to 279.5. CDO1 methylation was significantly different between cStage I and cStage II/III (P = 0.02). (ii) On the log-rank plot, the optimal cut-off value was determined to be 8.9; ESCC patients with high CDO1 methylation showed a significantly worse prognosis than those with low CDO1 methylation (P = 0.02). (iii) A multivariate Cox proportional hazards model identified only CDO1 hypermethylation as an independent prognostic factor (HR 2.00, CI 1.09-3.78, P = 0.03). (iv) CDO1 hypermethylation stratified ESCC patients' prognosis in cStage II/III for both neoadjuvant chemo(radio)therapy (NAC)-positive and NAC-negative cases. Moreover, the CDO1 methylation level was significantly lower in cases with Grade 2/3 than in those with Grade 0/1 (P = 0.02) among cStage II/III ESCC patients with NAC. Promoter DNA hypermethylation of CDO1 could be an independent prognostic factor in ESCC; it may also reflect NAC eradication of tumor cells in the primary tumors.

Homeobox-Only Protein Expression Is a Critical Prognostic Indicator of Pancreatic Neuroendocrine Tumor and Is Regulated by Promoter DNA Hypermethylation.

OBJECTIVES: We have identified homeobox-only protein (HOPX) as a tumor suppressor gene in various human cancer, and its expression was reduced by promoter DNA hypermethylation. Homeobox-only protein is strongly expressed on pancreatic islet cells; however, clinical relevance of HOPX expression has remained elusive in pancreatic neuroendocrine tumor (pNET). METHODS: We investigated 36 patients with pNET who undertook surgical resection between 1988 and 2012 for HOPX expression and DNA methylation to reveal its clinical significance. RESULTS: (1) Homeobox-only protein is strongly expressed on pancreatic islet cells by immunohistochemistry (IHC). Homeobox-only protein expression was recognized on pNET tumor cells for 1+ in 15, for 2+ in 16, and for 3+ in 5. (2) Homeobox-only protein IHC expression was significantly associated with prognosis (P = 0.03), and survival rate was 37.5%, 70.3%, and 100% in HOPX 1+, 2+, and 3+, respectively. (3) Promoter DNA methylation was quantitatively assessed, and HOPX hypermethylation is found in 6.3%, 11.8%, and 66.7% of G1/G2/G3 pNET, respectively (P = 0.02). (4) Multivariate Cox proportional hazards model identified HOPX IHC expression and HOPX promoter DNA hypermethylation as independent prognostic factors in pNET. CONCLUSIONS: Homeobox-only protein expression is a critical prognostic indicator of pNET, and its regulation may be made through promoter DNA methylation.

Vascular Endothelial Growth Factor Receptor Type 1 Signaling Prevents Delayed Wound Healing in Diabetes by Attenuating the Production of IL-1ß by Recruited Macrophages.

The persistence of proinflammatory macrophages, which are recruited to the granulation tissue, impairs the healing of diabetic wounds. Herein, we examined the role of vascular endothelial growth factor receptor type 1 (VEGFR1) signaling in streptozotocin (STZ)-induced diabetic wound healing. Angiogenesis, lymphangiogenesis, and the healing of full-thickness skin wounds were impaired in STZ-treated wild-type (WT) mice compared with vehicle-treated WT mice, with attenuated recruitment of VEGFR1-positive macrophages expressing vascular endothelial growth factor (VEGF)-A, VEGF-C, and VEGF-D to the wound granulation tissue. These phenomena were even more prevalent in STZ-treated VEGFR1 tyrosine kinase knockout mice (VEGFR1 TK(-/-) mice). STZ-treated WT mice, but not STZ-treated VEGFR1 TK(-/-) mice, showed accelerated wound healing when treated with placenta growth factor. Compared with that of STZ-treated WT mice, the wound granulation tissue of STZ-treated VEGFR1 TK(-/-) mice contained more VEGFR1-positive cells expressing IL-1ß [a classic (M1) activated macrophage marker] and fewer VEGFR1-positive cells expressing the mannose receptor [CD206; an alternatively activated (M2) macrophage marker]. Treatment of STZ-treated VEGFR1 TK(-/-) mice with an IL-1ß-neutralizing antibody restored impaired wound healing and angiogenesis/lymphangiogenesis and induced macrophages in the wound granulation tissue to switch to an M2 phenotype. Taken together, these results suggest that VEGFR1 signaling plays a role in regulating the balance between macrophage phenotypes in STZ-induced diabetic wounds, prevents impaired diabetic wound healing, and promotes angiogenesis/lymphangiogenesis.

Prognostic Significance of Promoter DNA Hypermethylation of cysteine dioxygenase 1 (CDO1) Gene in Primary Breast Cancer.

Using pharmacological unmasking microarray, we identified promoter DNA methylation of cysteine dioxygenase 1 (CDO1) gene in human cancer. In this study, we assessed the clinicopathological significance of CDO1 methylation in primary breast cancer (BC) with no prior chemotherapy. The CDO1 DNA methylation was quantified by TaqMan methylation specific PCR (Q-MSP) in 7 BC cell lines and 172 primary BC patients with no prior chemotherapy. Promoter DNA of the CDO1 gene was hypermethylated in 6 BC cell lines except SK-BR3, and CDO1 gene expression was all silenced at mRNA level in the 7 BC cell lines. Quantification of CDO1 methylation was developed using Q-MSP, and assessed in primary BC. Among the clinicopathologic factors, CDO1 methylation level was not statistically significantly associated with any prognostic factors. The log-rank plot analysis elucidated that the higher methylation the tumors harbored, the poorer prognosis the patients exhibited. Using the median value of 58.0 as a cut-off one, disease specific survival in BC patients with CDO1 hypermethylation showed significantly poorer prognosis than those with hypomethylation (p = 0.004). Multivariate Cox proportional hazards model identified that CDO1 hypermethylation was prognostic factor as well as Ki-67 and hormone receptor status. The most intriguingly, CDO1 hypermethylation was of robust prognostic relevance in triple negative BC (p = 0.007). Promoter DNA methylation of CDO1 gene was robust prognostic indicator in primary BC patients with no prior chemotherapy. Prognostic relevance of the CDO1 promoter DNA methylation is worthy of being paid attention in triple negative BC cancer.

Epigenetic regulation of ZEB1-RAB25/ESRP1 axis plays a critical role in phenylbutyrate treatment-resistant breast cancer.

Phenylbutyrate (PB) is a histone deacetylase antagonist that also exhibits antitumor activity. In this study, we used 7 breast cancer cell lines to identify biomarker candidates that predict PB sensitivity in breast cancer.Comprehensive gene expression profiles were compared using microarrays, and the importance of the identified genes to PB sensitivity was confirmed in gene transfection experiments. CRL and MDAMB453 cells were identified as PB-sensitive, while MDAMB231 cells were PB-resistant.RAB25 and ESRP1 were identified as key regulators of PB sensitivity, while ANKD1, ETS1, PTRF, IFI16 and KIAA1199 acted as PB resistance-related genes. Expression of these genes was dramatically altered by DNA demethylation treatments. RAB25 expression inhibited IFI16 and PTRF, while ESRP1 expression suppressed ANKRD1, ETS1, and KIAA1199. Both RAB25 and ESRP1 were suppressed by ZEB1, which was in turn regulated via epigenetic mechanisms. Thus, PB sensitivity is influenced by epigenetic expression alteration of ZEB1. The genes associated with PB sensitivity are downstream targets of ZEB1. Epigenetic regulation of ZEB1 may prove valuable as a critical biomarker for predicting resistance to breast cancer therapies.