Integrated profiling identifies DXS253E as a potential prognostic marker in colorectal cancer.

BACKGROUND: Increasing evidence suggests that DXS253E is critical for cancer development and progression, but the function and potential mechanism of DXS253E in colorectal cancer (CRC) remain largely unknown. In this study, we evaluated the clinical significance and explored the underlying mechanism of DXS253E in CRC. METHODS: DXS253E expression in cancer tissues was investigated using the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. The Kaplan-Meier plot was used to assess the prognosis of DXS253E. The cBioPortal, MethSurv, and Tumor Immune Estimation Resource (TIMER) databases were employed to analyze the mutation profile, methylation, and immune infiltration associated with DXS253E. The biological functions of DXS253E in CRC cells were determined by CCK-8 assay, plate cloning assay, Transwell assay, flow cytometry, lactate assay, western blot, and qRT-PCR. RESULTS: DXS253E was upregulated in CRC tissues and high DXS253E expression levels were correlated with poor survival in CRC patients. Our bioinformatics analyses showed that high DXS253E gene methylation levels were associated with the favorable prognosis of CRC patients. Furthermore, DXS253E levels were linked to the expression levels of several immunomodulatory genes and an abundance of immune cells. Mechanistically, the overexpression of DXS253E enhanced proliferation, migration, invasion, and the aerobic glycolysis of CRC cells through the AKT/mTOR pathway. CONCLUSIONS: We demonstrated that DXS253E functions as a potential role in CRC progression and may serve as an indicator of outcomes and a therapeutic target for regulating the AKT/mTOR pathway in CRC.

Tandem Mass Tag-Based Proteomic Profiling Identifies Biomarkers in Drainage Fluid for Early Detection of Anastomotic Leakage after Rectal Cancer Resection.

Anastomotic leakage (AL), one of the most severe complications in rectal surgery, is often diagnosed late because of the low specificity of the clinical symptoms and limitations of current clinical investigations. Identification of patients with early AL remains challenging. Here, we explored the protein expression profiles of AL patients to provide potential biomarkers to identify AL in patients who undergo surgery for rectal cancer. We screened differentially expressed proteins (DEPs) in drainage fluid from AL and non-AL patients using a tandem mass tag method. A total of 248 DEPs, including 98 upregulated and 150 downregulated proteins, were identified between AL and non-AL groups. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses suggested that DEPs were enriched in neutrophil degranulation, bacterial infection, proteolysis, hemostasis, and complement and coagulation cascades. The results of enzyme-linked immunosorbent assay validated that the expression of the top three upregulated DEPs, AMY2A, RETN, and CELA3A, was significantly increased in the drainage fluid of AL patients, compared with that of non-AL patients (AMY2A, P = 0.001; RETN, P < 0.0001; and CELA3A, P = 0.023). Thus, our findings provide several potential biomarkers for the early diagnosis of AL after rectal cancer resection.

WFDC3 inhibits tumor metastasis by promoting the ERß-mediated transcriptional repression of TGFBR1 in colorectal cancer.

Estrogen plays a protective role in colorectal cancer (CRC) and primarily functions through estrogen receptor ß (ERß). However, clinical strategies for CRC therapy associated with ERß are still under investigation. Our discoveries identified WFDC3 as a tumor suppressor that facilitates estrogen-induced inhibition of metastasis through the ERß/TGFBR1 signaling axis. WFDC3 interacts with ERß and increases its protein stability by inhibiting its proteasome-dependent degradation. WFDC3 represses TGFBR1 expression through ERß-mediated transcription. Blocking TGFß signaling with galunisertib, a drug used in clinical trials that targets TGFBR1, impaired the migration of CRC cells induced by WFDC3 depletion. Moreover, there was clinical significance to WFDC3 in CRC, as CRC patients with high WFDC3 expression in tumor cells had favorable prognoses. Therefore, this work suggests that WFDC3 could be an indicator for therapies targeting the estrogen/ERß pathway in CRC patients.

Genome-wide transcriptomics and copy number profiling identify patient-specific CNV-lncRNA-mRNA regulatory triplets in colorectal cancer.

Screening cancer genomes has provided an in-depth characterization of genetic variants such as copy number variations (CNVs) and gene expression changes of non-coding transcripts. Single-dimensional experiments are often designed to differentiate a patient cohort into various sets with the aim of identifying molecular changes among groups; however, this may be inadequate to decipher the causal relationship between molecular signatures in individual patients. To overcome this challenge with respect to personalized medicine, we implemented a patient-specific multi-dimensional integrative approach to uncover coherent signals from multiple independent platforms. In particular, we focused on the consistent gene dosage effects of CNVs for both mRNA and long non-coding RNA (lncRNA) expression in nine colorectal cancer patients. We identified 511 CNV-lncRNA-mRNA regulatory triplets associated with CNVs and aberrant expression of both mRNAs and lncRNAs. By filtering out inconsistent changes among CNVs, mRNAs, and lncRNAs, we further characterized 165 coherent motifs associated with 56 genes. In total, 108 motifs were linked with 31 copy number gains, 44 upregulated lncRNAs, and 45 upregulated mRNAs. Another 57 coherent downregulated motifs were also collected. We discuss how for many of these CNV-lncRNA-mRNA regulatory triplets, their clinical impact remains to be explored, including survival time, microsatellite instability, tumor stage, and primary tumor sites. By validating two example CNV-lncRNA-mRNA triplets with up- and down-regulation, we confirmed that individual variations in multiple dimensions are a robust tool to identify reliable molecular signals for personalized medicine. In summary, we utilized a patient-specific computational pipeline to explore the consistent CNV-driven motifs consisting of lncRNAs and mRNAs. We also identified LSM14B as a potential promoter in colorectal cancer progression, suggesting that it may serve as a target for colorectal cancer treatment.

Lymph node yield less than 12 is not a poor predictor of survival in locally advanced rectal cancer after laparoscopic TME following neoadjuvant chemoradiotherapy.

Purpose: Previous studies have confirmed that neoadjuvant chemoradiotherapy (nCRT) may reduce the number of lymph nodes retrieved in rectal cancer. However, it is still controversial whether it is necessary to harvest at least 12 lymph nodes for locally advanced rectal cancer (LARC) patients who underwent nCRT regardless of open or laparoscopic surgery. This study was designed to evaluate the relationship between lymph node yield (LNY) and survival in LARC patients who underwent laparoscopic TME following nCRT. Methods: Patients with LARC who underwent nCRT followed by laparoscopic TME were retrospectively analyzed. The relationship between LNY and survival of patients was evaluated, and the related factors affecting LNY were explored. To further eliminate the influence of imbalance of clinicopathological features on prognosis between groups, propensity score matching was conducted. Results: A total of 257 consecutive patients were included in our study. The median number of LNY was 10 (7 to 13) in the total cohort. There were 98 (38.1%) patients with 12 or more lymph nodes harvested (LNY ≥12 group), and 159 (61.9%) patients with fewer than 12 lymph nodes retrieved (LNY <12 group). There was nearly no significant difference between the two groups in clinicopathologic characteristics and surgical outcomes except that the age of LNY <12 group was older (P<0.001), and LNY <12 group tended to have more TRG 0 cases (P<0.060). However, after matching, when 87 pairs of patients obtained, the clinicopathological features were almost balanced between the two groups. After a median follow-up of 65 (54 to 75) months, the 5-year OS was 83.9% for the LNY ≥12 group and 83.6% for the LNY <12 group (P=0.893), the 5-year DFS was 78.8% and 73.4%, respectively (P=0.621). Multivariate analysis showed that only patient age, TRG score and ypN stage were independent factors affecting the number of LNY (all P<0.05). However, no association was found between LNY and laparoscopic surgery-related factors. Conclusions: For LARC patients who underwent nCRT followed by laparoscopic TME, the number of LNY less than 12 has not been proved to be an adverse predictor for long-term survival. There was no correlation between LNY and laparoscopic surgery-related factors.

Patterns and predictors of recurrence after laparoscopic resection of rectal cancer.

Purpose: This study was designed to evaluate the patterns and predictors of recurrence in patients who underwent laparoscopic resection of rectal cancer. Methods: Patients with rectal cancer receiving laparoscopic resection between April 2009 and March 2016 were retrospectively analyzed. The association of recurrence with clinicopathological characteristics was evaluated using multivariate analyses. Results: A total of 405 consecutive patients were included in our study. Within a median follow-up time of 62 months, 77 patients (19.0%) experienced disease recurrence: 10 (2.5%) had locoregional recurrence (LR), 61 (15.1%) had distant metastasis (DM), and 6 (1.5%) developed LR and DM synchronously. The lung was the most common site of metastasis. Multivariate analyses indicated that involved circumferential resection margin (CRM) was the only independent predictor for LR (OR=13.708, 95% CI 3.478-54.026, P<0.001), whereas elevated baseline level of CA19-9 (OR=3.299, 95% CI 1.461-7.449, P=0.032), advanced pN stage (OR=2.292, 95% CI 1.177-4.462, P=0.015) and harvested lymph nodes less than 12 (OR=2.418, 95% CI 1.245-4.695, P=0.009) were independently associated with DM. Patients receiving salvage surgery showed superior 3-year survival compared with palliative treatment after relapse (90.9% vs. 20.5%; P=0.017). The estimated 5-year DFS and CSS for the entire cohort was 80.2% and 83.1%, respectively. Conclusions: DM was more common than LR after laparoscopic resection of rectal cancer, and there were several clinicopathological factors related to LR and DM. Involved CRM and suboptimal lymph node yield were adverse surgery-related factors of tumor recurrence, which should be paid more attention to during the operation.

A stop-gain mutation in GXYLT1 promotes metastasis of colorectal cancer via the MAPK pathway.

Genomic instability plays a key role in the initiation and progression of colorectal cancer (CRC). Although cancer driver genes in CRC have been well characterized, identifying novel genes associated with carcinogenesis and treatment remains challenging because of tumor heterogeneity. Here, we analyzed the genomic alterations of 45 samples from CRC patients in northern China by whole-exome sequencing. In addition to the identification of six well-known CRC driver genes (APC, TP53, KRAS, FBXW7, PIK3CA, and PABPC), two tumor-related genes (MTCH2 and HSPA6) were detected, along with RRP7A and GXYLT1, which have not been previously linked to cancer. GXYLT1 was mutated in 40% (18/45) of the samples in our cohort. Functionally, GXYLT1 promoted migration and invasion in vitro and metastasis in vivo, while the GXYLT1S212* mutant induced significantly greater effect. Furthermore, both GXYLT1 and GXYLT1S212* interacted with ERK2. GXYLT1 induced metastasis via a mechanism involving the Notch and MAPK pathways, whereas the GXYLT1S212* mutant mainly promoted metastasis by activating the MAPK pathway. We propose that GXYLT1 acts as a novel metastasis-associated driver gene and GXYLT1S212* might serve as a potential indicator for therapies targeting the MAPK pathway in CRC.

Downregulation of STK25 promotes autophagy via the Janus kinase 2/signal transducer and activator of transcription 3 pathway in colorectal cancer.

Autophagy plays a crucial role in colorectal cancer (CRC) development. Our previous study suggested that serine/threonine protein kinase 25 (STK25) regulates aerobic glycolysis in CRC cells. Glycolysis modulates cellular autophagy during tumor growth; however, the role of STK25 in autophagy remains unclear. In this study, we found that STK25 expression was decreased in CRC tissues and CRC patients with high STK25 expression had a favorable prognosis. Functional assays suggested that STK25 inhibition promoted autophagy in CRC cells. Overexpression of STK25 exhibited the opposite effects. Moreover, the results of western blot demonstrated that silencing STK25 induced autophagy by activating the JAK2/STAT3 pathway. Therefore, STK25 could be a potential indicator for therapies targeting the JAK2/STAT3 pathway in CRC.

Monitoring Pre- and Post-Operative Immune Alterations in Patients With Locoregional Colorectal Cancer Who Underwent Laparoscopy by Single-Cell Mass Cytometry.

Surgical excision is currently the principal therapy for locoregional colorectal cancer (CRC). However, surgical trauma leads to controlled tissue damage, causing profound alterations in host immunity and, in turn, affecting post-operative outcomes. Surgery-induced immune alterations in CRC remain poorly defined. Here, single-cell mass cytometry was applied to serial blood samples collected pre-operatively, and on days 1, 3, and 7 post-operatively from 24 patients who underwent laparoscopic surgical resection of CRC to comprehensively monitor the perioperative phenotypic alterations in immune cells and dynamics of immune response. Characterization of immune cell subsets revealed that the post-operative immune response is broad but predominantly suppressive, supported by the decreases in total frequencies of circulating T cells and natural killer (NK) cells, as well as decreased HLA-DR expression on circulating monocytes. The proportion of T cells significantly decreased on day 1 and recovered to the pre-surgical level on day 3 after surgery. The frequency of monocytes was significantly elevated on day 1 after surgery and declined to baseline level on day 3. NK cells temporarily contracted on post-operative day 3. T cells, monocytes, DCs, NK cells, and B cells were partitioned into phenotypically different single-cell clusters. The dynamics of single-cell clusters were different from those of the bulk lineages. T cell clusters in the same response phase fluctuate inconsistently during the perioperative period. Comparing to the baseline levels, the frequencies of CD11b(+)CD33(+)CD14(+)CD16(-) classical monocytes expanded followed by contraction, whereas CD11b(+)CD33(+)CD14(high)CD16(low) intermediate monocytes remained unchanged; HLA-DR expression in monocytes were significantly reduced; the frequencies of intermediate CD56(bright)CD16(+) NK cell subsets increased; and the percentage of memory B lymphocytes were elevated after surgery. Post-operative pro- and anti-inflammatory cytokines were both altered. Furthermore, perioperative immune perturbations in some of the cell subsets were unrecovered within seven days after surgery. Chronological monitoring major immune lineages provided an overview of surgery-caused alterations, including cell augments and contractions and precisely timed changes in immune cell distribution in both innate and adaptive compartments, providing evidence for the interaction between tumor resection and immune modulation.

Genome-wide circular RNA (circRNA) and mRNA profiling identify a circMET-miR-410-3p regulatory motif for cell growth in colorectal cancer.

Circular RNA (circRNA) is a non-coding RNA molecule that lacks polyadenylated tails and is highly stable, abundant, and conserved in human cells. CircRNAs can serve as a competing endogenous RNA (ceRNA) to sponge microRNAs (miRNA) and block their effects on target mRNA expression. CircRNAs also have possible relevance to cancer and therefore may be considered as ideal biomarkers for monitoring cancer progression. Of the about 300,000 predicted human circRNAs, only a few have validated biological functions related to cancer. To better understand the ceRNA role of circRNAs in colorectal cancer (CRC), we performed genome-wide circRNA-based RNA-sequencing (RNA-Seq) on nine CRC tumor samples and their paired histologically normal adjacent tissue samples. By profiling the mRNA expression in the same patients, we further explored the expression correlation between circRNAs and mRNAs generated from the same parental gene. Focusing on the concordant differential expression between circRNAs and mRNAs, we substantially reduced the regulatory noise. In total, we identified 694 circRNA-mRNA pairs that were consistently up or downregulated between tumor and normal tissues. These 694 circRNA-mRNA pairs are from 182 protein-coding genes associated with hormone responses and chemotaxis. Of these 182 genes, 43 are downstream targets of three highly conserved miRNAs (miR-410-3p, miR-135a, and miR-30a). Interestingly, these 43 genes are highly mutated in another cohort from eight independent CRC studies, which have significant effects on patient survival time. Focusing on miR-410-3p and its target oncogene MET, we experimentally validated the ceRNA regulatory motif of circMET. Notably, circMET is substantially upregulated in CRC cell lines and could promote cell proliferation and growth. By confirming the regulatory relationship between miR-410-3p and circMET, we propose a new mechanism for the observed sustained activation of MET in CRC. In conclusion, our work identifies a novel regulatory role of circMET and provides a potential diagnostic biomarker for CRC.

Adjuvant chemotherapy is an additional option for locally advanced gastric cancer after radical gastrectomy with D2 lymphadenectomy: a retrospective control study.

BACKGROUND: This study compared the long-term efficacy of different durations of adjuvant chemotherapy for patients with gastric cancer after radical gastrectomy with D2 lymphadenectomy. METHODS: We retrospectively identified 428 patients with stage II-III gastric cancer who underwent D2 gastrectomy between 2009 and 2016. Patients were divided into four groups according to the duration of adjuvant chemotherapy, including 0 week (no adjuvant, group A), 20 to 24 weeks (completed 7-8 cycles every 3 weeks or 10-12 cycles every 2 weeks, group B), and 12 to18 weeks (completed 4-6 cycles every 3 weeks or 6-9 cycles every 2 weeks, group C), and less than 12 weeks (received up to 3 cycles every 3 weeks or 5 cycles every 2 weeks, group D). The chemotherapy regimens included XELOX, SOX, and FOLFOX. 5-year overall survival (OS) and disease-free survival (DFS) were analyzed. RESULTS: The 5-year OS rates for groups A, B, C, and D were 52.3, 73.7, 72.0, and 53.3%, respectively, and the 5-year DFS rates were 50.0, 68.0, 65.4, and 50.0%, respectively. OS and DFS were higher in group B than in groups A and D. Similarly, patients in group C were more likely to have higher OS and DFS than those in groups A and D. Meanwhile, there were no significant differences in OS and DFS between groups B and C. The multivariate analysis confirmed with high statistical significance the efficacy of complete courses of adjuvant chemotherapy, and, among them, the similar impact of 4-6/6-9 and 7-8/10-12 cycles, resulting in similar HRs vs Group A (0.52 and 0.42, respectively). CONCLUSIONS: To reduce toxicity and maintain efficacy, XELOX or SOX chemotherapy regimens administered for 4-6 cycles every 3 weeks or FOLFOX regimen for 6-9 cycles every 2 weeks might be a favorable option for patients with stage II-III gastric cancer after D2 gastrectomy. Prospective multicenter clinical trials with adequate sample sizes are necessary to verify these findings.

Quadruple-editing of the MAPK and PI3K pathways effectively blocks the progression of KRAS-mutated colorectal cancer cells.

Mutated KRAS promotes the activation of the MAPK pathway and the progression of colorectal cancer (CRC) cells. Aberrant activation of the PI3K pathway strongly attenuates the efficacy of MAPK suppression in KRAS-mutated CRC. The development of a novel strategy targeting a dual pathway is therefore highly essential for the therapy of KRAS-mutated CRC. In this study, a quadruple-depleting system for the KRAS, MEK1, PIK3CA, and MTOR genes based on CRISPR/SaCas9 was developed. Adenovirus serotype 5 (ADV5) was integrated with two engineered proteins, an adaptor and a protector, to form ADV-protein complex (APC) for systemic delivery of the CRISPR system. Quadruple-editing could significantly inhibit the MAPK and PI3K pathways in CRC cells with oncogenic mutations of KRAS and PIK3CA or with KRAS mutation and compensated PI3K activation. Compared with MEK and PI3K/MTOR inhibitors, quadruple-editing induced more significant survival inhibition on primary CRC cells with oncogenic mutations of KRAS and PIK3CA. The adaptor specifically targeting EpCAM and the hexon-shielding protector could dramatically enhance ADV5 infection efficiency to CRC cells and significantly reduce off-targeting tropisms to many organs except the colon. Moreover, quadruple-editing intravenously delivered by APC significantly blocked the dual pathway and tumor growth of KRAS-mutated CRC cells, without influencing normal tissues in cell- and patient-derived xenograft models. Therefore, APC-delivered quadruple-editing of the MAPK and PI3K pathways shows a promising therapeutic potential for KRAS-mutated CRC.

Genomic profiling of colorectal cancer with isolated lung metastasis.

BACKGROUND: Metastasis is a major cause of failed colorectal cancer (CRC) treatment. While lung metastasis (LM) is observed in 10-15% of patients with CRC, the genetic mechanisms that cause CRC to metastasize to the lung remain unclear. METHODS: In this study, we employed whole exome sequencing (WES) of primary CRC tumors and matched isolated LM lesions to compare their genomic profiles. Comprehensive genomic analyses of five freshly frozen primary tumor lesions, five paired LM lesions, and matched non-cancerous tissues was achieved by WES. RESULTS: An integrated analysis of somatic mutations, somatic copy number alterations, and clonal structures revealed that genomic alterations were present in primary and metastatic CRCs with various levels of discordance, indicating substantial levels of intertumor heterogeneity. Moreover, our results suggest that the founder clone of the primary tumor was responsible for the formation of the metastatic lesion. Additionally, only a few metastasis-specific mutations were identified, suggesting that LM-promoting mutations might be pre-existing in primary tumors. CONCLUSIONS: Primary and metastatic CRC show intertumor heterogeneity; however, both lesions were founded by the same clone. These results indicate that malignant clones contributing to disease progression should be identified during the genetic prognosis of cancer metastasis.

Phenotype molding of T cells in colorectal cancer by single-cell analysis.

The majority of patients with microsatellite stable (MSS) colorectal cancer (CRC) do not benefit from the immunotherapies directed at rescuing T-cell functions. Therefore, complete understanding of T-cell phenotypes and functional status in the CRC microenvironment is desirable. Here, we applied single-cell mass cytometry to mold the T-cell phenotype in 18 patients with MSS CRC for better understanding of CRC as a systemic disease and to search for tumor-driven T-cell profile changes. We show interpatient and intrapatient phenotypic diversity of T-cell subsets. We revealed increased immunosuppressive/exhausted T-cell phenotypes at tumor lesions. CD8+ CD28- immunosenescent T cells with impaired proliferation capacity dominate the T-cell compartment. As per the transcriptome and quantitative real time-PCR analysis, the accumulation of immunosuppressive cells is driven by the tumor microenvironment. T-cell profiles are similar between patients at early and late stages, indicating that the immunosuppressive microenvironment is formulated early during CRC development. Mapping of T-cell infiltration and understanding of the mechanisms underlying their regulation may provide valuable information to boost the immune response in patients with MSS CRC.

Clonality and heterogeneity of metachronous colorectal cancer.

Patients with metachronous colorectal cancer (CRC) have been diagnosed with primary CRC more than once. Given that the genetic and microenvironment is the same in these cases, metachronous CRC is an important model for studying colorectal tumorigenesis. We performed whole exome sequencing of seven freshly frozen tumors from three patients with metachronous CRC and compared their genetic profiles. In patients with metachronous tumors of distinct genetic origins, 3.74% and 0.20% of genes were ubiquitously mutated and candidate cancer genes mutated at different sites. Tumors from the same patients were clonally unrelated, and thus druggable genes differed. In contrast, in a patient with metachronous tumors of a common genetic origin, the ubiquitously mutated genes were 61.02%, with ubiquitously mutated genes and candidate cancer genes all mutated at the same sites, tumors were clonally related, and some druggable genes were the same. Therefore, two different clonal relationships between metachronous tumors exist in CRC, one is monoclonal and the other is polyclonal. Our findings may help to advance understanding of the differences in metachronous CRCs and the genetic mechanisms of which they originate, and provide new avenues for CRC treatment.

KIF14 promotes cell proliferation via activation of Akt and is directly targeted by miR-200c in colorectal cancer.

As a mitotic kinesin, kinesin family member 14 (KIF14) has been reported to serve oncogenic roles in a variety of malignancies; however, its functional role and regulatory mechanisms in colorectal cancer (CRC) remain unclear. In the present study, KIF14 was observed to be markedly overexpressed in CRC, and this upregulation was associated with tumor size and marker of proliferation Ki-67 immunostaining scores. Gain- and loss-of-function experiments were applied to identify the function of KIF14 in CRC progression. In vitro and in vivo assays revealed that KIF14 promoted CRC cell proliferation and accelerated the cell cycle via activation of protein kinase B. In addition, the present study investigated the potential mechanisms underlying KIF14 overexpression in CRC. Bioinformatics analyses and validation experiments, including reverse transcription-quantitative polymerase chain reaction, western blotting and a Dual-Luciferase reporter assay, demonstrated that, in addition to genomic amplification and transcriptional activation, KIF14 was regulated by microRNA (miR)-200c at the post-transcriptional level. Rescue experiments further demonstrated that decreased miR-200c expression could facilitate KIF14 to exert its pro-proliferative role. The expression of miR-200c was negatively correlated with KIF14 in CRC specimens. Collectively, the findings of the present study demonstrated the oncogenic role of KIF14 in colorectal tumorigenesis, and also revealed a complexity of regulatory mechanisms mediating KIF14 overexpression, which may provide insight for developing novel treatments for patients with CRC.

STK25-induced inhibition of aerobic glycolysis via GOLPH3-mTOR pathway suppresses cell proliferation in colorectal cancer.

BACKGROUND: Serine/threonine protein kinase 25 (STK25) is critical in regulating whole-body glucose and insulin homeostasis and the accumulation of ectopic lipids. The Warburg effect, also known as aerobic glycolysis, is an essential metabolic characteristic of cancer cells. However, the effects of STK25 on aerobic glycolysis of cancer cells remain unexplored. The aim of this study is to investigate the role of STK25 in colorectal cancer (CRC) and to elucidate the underlying mechanisms. METHODS: The influences of STK25 on the cell proliferation were evaluated by MTT and colony formation assays. The roles of STK25 in aerobic glycolysis were determined by glucose uptake and lactate production assays. The interaction between STK25 and GOLPH3 was detected by co-immunoprecipitation, GST pull-down, and His-tag pull-down assays. Western blot was used to measure the expression of glycolytic genes, and the status of kinases in mTOR pathway. Moreover, a xenograft mouse model was used to investigate the effects of STK25 in vivo. The prognostic significance of STK25 was analyzed using public CRC datasets by a log-rank test. RESULTS: STK25 suppressed proliferation, glycolysis and glycolytic gene expression in CRC cells. STK25 interacted with GOLPH3 and mediated glycolysis through GOLPH3-regulated mTOR signaling. Consistent with these observations, silencing of STK25 promoted tumor growth and glycolytic gene expression in an in vivo xenograft mouse model. Moreover, high levels of STK25 correlated with favorable prognosis in patients with CRC. CONCLUSIONS: Our results demonstrated that STK25 negatively regulates the proliferation and glycolysis via GOLPH3-dependent mTOR signaling. Accordingly, STK25 could be a potential therapeutic target for the treatment of CRC.

Comparison of EpCAMhighCD44+ cancer stem cells with EpCAMhighCD44- tumor cells in colon cancer by single-cell sequencing.

Cancer stem cells (CSCs) are considered to be responsible for tumorigenesis and cancer relapse. EpCAMhighCD44+ tumor cells are putative colorectal CSCs that express high levels of stem cell genes, while the EpCAMhighCD44- population mostly contains differentiated tumor cells (DTCs). This study aims to determine whether single CSC (EpCAMhighCD44+) and DTC (EpCAMhighCD44-) can be distinguished in terms of somatic copy number alterations (SCNAs). We applied fluorescence-activated cell sorting to isolate the CD45-EpCAMhighCD44+ and CD45-EpCAMhighCD44- populations from two primary colon tumors, on which low-coverage single-cell whole-genome sequencing (WGS) was then performed ∼0.1x depth. We compared the SCNAs of the CSCs and DTCs at single-cell resolution. In total, 47 qualified single cells of the two populations underwent WGS. The single-cell SCNA profiles showed that there were obvious SCNAs in both the CSCs and DTCs of each patient, and each patient had a specific copy number alteration pattern. Hierarchical clustering and correlation analysis both showed that the SCNA profiles of CSCs and DTCs from the same patient had similar SCNA pattern, while there were regional differences in the CSCs and DTCs in certain patient. SCNAs of CSCs in the same patient were highly reproducible. Our data suggest that major SCNAs occurred at an early stage and were inherited steadily. The similarity of ubiquitous SCNAs between the CSCs and DTCs might have arisen from lineage differentiation. CSCs from the same patient had reproducible SCNA profiles, indicating that gain or loss in certain chromosome is required for colon cancer development.

Whole exome sequencing reveals intertumor heterogeneity and distinct genetic origins of sporadic synchronous colorectal cancer.

Sporadic synchronous colorectal cancer (CRC) refers to more than one primary tumor detected in a single patient at the time of the first diagnosis without predisposition of cancer development. Given the same genetic and microenvironment they raise, sporadic synchronous CRC is a unique model to study CRC tumorigenesis. We performed whole exome sequencing in 32 fresh frozen tumor lesions from 15 patients with sporadic synchronous CRC to compare their genetic alterations. This approach identified ubiquitously mutated genes in the range from 0.34% to 4.22% and from 0.8% to 7.0% in non-hypermutated tumors and hypermutated tumors, respectively, in a single patient. We show that both ubiquitously mutated genes and candidate cancer genes from different tumors in the same patient mutated at different sites. Consistently, obvious differences in somatic copy number variations (SCNV) were found in most patients with non-hypermutated tumor lesions, which had ubiquitous copy number amplification rates ranging from 0% to 8.8% and ubiquitous copy number deletion rates ranging from 0% to 8.2%. Hypermutated lesions were nearly diploid with 0% to 18.8% common copy number aberrations. Accordingly, clonal structures, altered signaling pathways and druggable genes in a single patient with synchronous CRC varied significantly. Taken together, the disparate SCNVs and mutations in synchronous CRC supported the field effect theory of tumorigenesis. Moreover, the intertumor heterogeneity of synchronous CRCs implies that analysis of all tumor lesions from the same patient is necessary for appropriate clinical treatment decisions.

Clinical significance of circulating immune cells in left- and right-sided colon cancer.

BACKGROUND: Left-sided and right-sided colon cancers (LCCs and RCCs, respectively) differ in their epidemiology, pathogenesis, genetic and epigenetic alterations, molecular pathways and prognosis. Notably, immune response gene expression profiles have been shown to differ between patients with LCC and patients with RCC. The immune system plays an important role in tumor immunosurveillance, and there is increasing evidence that peripheral blood immune cells have a profound influence on tumor prognosis. This study aimed to determine the clinical significance of circulating immune cells with respect to colon tumor locations. METHODS: Different types of circulating immune cells were separated and analysed based on their surface markers by flow cytometry. We compared the numbers of dendritic cells (DCs) and T cell subsets in the peripheral blood of 94 patients with RCC or LCC and analysed the proportions of these immune cells in relation to tumor stage, tumor differentiation and lymphatic metastasis. RESULTS: We show that at later tumor stages, patients with LCC had higher levels of circulating myeloid DCs (P = 0.049) and plasmacytoid DCs (P = 0.018) than patients with RCC. In poorly differentiated tumors, LCC patients had significantly higher amount of plasmacytoid DCs (P = 0.036), CD4+ memory T (Tm) cells (P = 0.012), CD4+ T cells (P = 0.028), Tm cells (P = 0.014), and regulatory T cells (P = 0.001) than RCC patients. The levels of circulating CD4+ T cells, Tm cells and CD4+ Tm cells were significantly elevated at later stages in patients with LCC or RCC, while these cells decreased in poorly differentiated tumors in patients with RCC. Moreover, CD4+ Tm cell and CD4+ T cell levels are significantly associated with lymph node metastasis in patients with LCC and RCC. DISCUSSION: Circulating immune cells were associated with tumor location, tumor stage and tumor differentiation, and can be used to predict lymphatic metastasis in patients with colon cancer. This variation in systemic immunity could contribute to the differential prognosis of patients with colon cancer.