Oxidative Damage in Sporadic Colorectal Cancer: Molecular Mapping of Base Excision Repair Glycosylases MUTYH and hOGG1 in Colorectal Cancer Patients

Oxidative stress, oxidative DNA damage and resulting mutations play a role in colorectal carcinogenesis. Impaired equilibrium between DNA damage formation, antioxidant status, and DNA repair capacity is responsible for the accumulation of genetic mutations and genomic instability. The lesion-specific DNA glycosylases, e.g., hOGG1 and MUTYH, initiate the repair of oxidative DNA damage. Hereditary syndromes (MUTYH-associated polyposis, NTHL1-associated tumor syndrome) with germline mutations causing a loss-of-function in base excision repair glycosylases, serve as straight forward evidence on the role of oxidative DNA damage and its repair. Altered or inhibited function of above glycosylases result in an accumulation of oxidative DNA damage and contribute to the adenoma-adenocarcinoma transition. Oxidative DNA damage, unless repaired, often gives rise G:C > T:A mutations in tumor suppressor genes and proto-oncogenes with subsequent occurrence of chromosomal copy-neutral loss of heterozygosity. For instance, G>T transversions in position c.34 of a KRAS gene serves as a pre-screening tool for MUTYH-associated polyposis diagnosis. Since sporadic colorectal cancer represents more complex and heterogenous disease, the situation is more complicated. In the present study we focused on the roles of base excision repair glycosylases (hOGG1, MUTYH) in colorectal cancer patients by investigating tumor and adjacent mucosa tissues. Although we found downregulation of both glycosylases and significantly lower expression of hOGG1 in tumor tissues, accompanied with G>T mutations in KRAS gene, oxidative DNA damage and its repair cannot solely explain the onset of sporadic colorectal cancer. In this respect, other factors (especially microenvironment) per se or in combination with oxidative DNA damage warrant further attention. Base excision repair characteristics determined in colorectal cancer tissues and their association with disease prognosis have been discussed as well.     

Polymorphisms in XPD and ERCC1 Associated with Colorectal Cancer Outcome

Using the comprehensive approach to selecting polymorphisms to date, we sought to examine whether recurrence in colorectal cancer was associated with inherited variation in three genes involved in DNA repair and cell proliferation. Three polymorphisms, which are excision repair cross-complementation 1 (ERCC1), xeroderma pigmentosum group D (XPD) and epidermal growth factor receptor (EGFR), were assessed in 257 postoperative stage II/III CRC patients with 5-fluorouracial chemotherapy in Taiwan. In addition, the correlations between genetic polymorphisms and patients’ clinicopathological features were investigated. Genotypes of XPD codon751 A/A and ERCC1 codon118 T/T were associated with regional recurrence in a statistically significant way (p = 0.018). Patients who carried XPD AA and ERCC1 TT genotypes demonstrated a significantly greater regional recurrence risk (OR = 5.625, 95% CI, 1.557–20.32). Inherited variation in XPD and ERCC1 was associated with outcome in patients with colorectal cancer in Taiwan. As the significant association of single-nucleotide polymorphisms has not been studied previously in colorectal cancer, these findings suggest novel sites of variation, in part explaining the range of treatment responses seen in this disease.     

Galanin Receptors (GalR1, GalR2, and GalR3) Expression in Colorectal Cancer Tissue and Correlations to the Overall Survival and Poor Prognosis of CRC Patients

Colorectal cancer (CRC) is the second most common cause of cancer in women and the third in men. The postoperative pathomorphological evaluation of patients with CRC is extremely important for future therapeutic decisions. Although our previous studies demonstrated high galanin (GAL) presence within tumor tissue and an elevated concentration of GAL in the serum of CRC patients, to date, there is a lack of data regarding GAL receptor (GalR) protein expression in CRC cells. Therefore, the aim of this study was to evaluate the presence of all three types of GalRs (GalR1, GalR2 and GalR3) within epithelial cells of the human colon and CRC tissue with the use of the immunohistochemical method and to correlate the results with the clinical-pathological data. We found stronger immunoreactivity of GalR1 and GalR3 in CRC cells compared to epithelial cells of the unchanged mucosa of the large intestine. No differences in the GalR2 protein immunoreactivity between the studied tissues were noted. We also found that the increased immunoexpression of the GalR3 in CRC tissue correlated with the better prognosis and longer survival (p < 0.0079) of CRC patients (n = 55). The obtained results suggest that GalR3 may play the role of a prognostic factor for CRC patients. Based on data from the TCGA-COAD project deposited in the GDC Data Portal, we also found that GalR mRNA in cancer samples and the adjacent normal tissue did not correlate with immunoexpression of the GalR proteins in CRC cells and epithelial cells of the unchanged mucosa.     

CD40 Pathway and IL-2 Expression Mediate the Differential Outcome of Colorectal Cancer Patients with Different CSF1R c.1085 Genotypes

Colony-stimulating factor 1 receptor (CSF-1R) acts as the receptor for colony stimulating factor 1, a cytokine that controls the production, differentiation, and function of macrophages. Prior studies showed cancer patients harboring germline CSF1R c.1085A>G genetic variant had better survival. Here, primary tumor samples from a stage III colorectal cancer (CRC) cohort were analyzed by a targeted gene expression assay containing 395 immune-related genes to study the immune mechanism underlying the different outcomes. CRC patients with CSF1R c.1085 genotype A_G had a better disease-free and overall survival than those with CSF1R genotype A_A. Compared to the group of patients without CSF1R variant, higher CD40LG expression, a surface marker of T cells, was found in the tumor tissues of patients with CSF1R c.1085 variant. In parallel with the higher CD40LG gene expression, immunofluorescent staining also showed more CD3⁺CD40L⁺ T cell infiltrates in tumors with CSF1R c.1085 genotype A_G. Moreover, higher IL-2 expression, known to be regulated by CD40 pathway, was also observed in tumors with CSF1R c.1085 genotype A_G than genotype A_A. Higher IL-2 expression generated by the interaction of CD40 ligand and CD40 between T cells and macrophages with CSF1R c.1085A>G variant is the potential mechanism explaining the different outcomes.     

Prognostic Impact and Functional Annotations of KIF11 and KIF14 Expression in Patients with Colorectal Cancer

Genomic instability (GIN) has an important contribution to the pathology of colorectal cancer (CRC). Therefore, we selected mitosis and cytokinesis kinesins, KIF11 and KIF14, as factors of potential clinical and functional value in CRC, as their aberrant expression has been suspected to underlie GIN. We examined the expression and the prognostic and biological significance of KIF11 and KIF14 in CRC via in-house immunohistochemistry on tissue microarrays, public mRNA expression datasets, as well as bioinformatics tools. We found that KIF11 and KIF14 expression, at both the protein and mRNA level, was markedly altered in cancer tissues compared to respective controls, which was reflected in the clinical outcome of CRC patients. Specifically, we provide the first evidence that KIF11 protein and mRNA, KIF14 mRNA, as well as both proteins together, can significantly discriminate between CRC patients with better and worse overall survival independently of other relevant clinical risk factors. The negative prognostic factors for OS were high KIF11 protein, high KIF11 protein + low KIF14 protein, low KIF11 mRNA and low KIF14 mRNA. Functional enrichment analysis revealed that the gene sets related to the cell cycle, DNA replication, DNA repair and recombination, among others, were positively associated with KIF11 or KIF14 expression in CRC tissues. In TCGA cohort, the positive correlations between several measures related to GIN and the expression of KIFs were also demonstrated. In conclusion, our results suggest that CRC patients can be stratified into distinct risk categories by biological and molecular determinants, such as KIF11 and KIF14 expression and, mechanistically, this is likely attributable to their role in maintaining genome integrity.     

NAD(P)H:Quinone Oxidoreductase 1 (NQO1) P187S Polymorphism and Prostate Cancer Risk in Caucasians

NAD(P)H:quinone oxidoreductase 1 (NQO1) catalyses the reduction of quinoid compounds to hydroquinones, preventing the generation of free radicals and reactive oxygen. A “C” to “T” transversion at position 609 of NQO1, leading to a nonsynonymous amino acid change (Pro187Ser, P187S), results in an altered enzyme activity. No NQO1 protein activity was detected in NQO1 ⁶⁰⁹TT genotype, and low to intermediate activity was detected in NQO1 ⁶⁰⁹CT genotype compared with ⁶⁰⁹CC genotype. Thus, this polymorphism may result in altered cancer predisposition. For prostate cancer, only sparse data are available. We therefore analyzed the distribution of the NQO1 P187S SNP (single nucleotide polymorphism) in prostate cancer patients and a healthy control group. Allelic variants were determined using RFLP analysis. Overall, 232 patients without any malignancy and 119 consecutive prostate cancer patients were investigated. The genotype distribution in our cohorts followed the Hardy–Weinberg equilibrium in cases and controls. The distribution of the NQO1 codon 187 SNP did not differ significantly between prostate cancer patients and the control group (p = 0.242). There was also no association between the allelic variants and stage or Gleason score of the tumors. The NQO1 P187S SNP was not significantly associated with an increased prostate cancer risk in our cohorts. The SNP has also no influence on histopathological characteristics of the tumors. A combined analysis of all available data from published European studies also showed no significant differences in the genotype distribution between controls and prostate cancer patients. Our data suggest a minor role of the NQO1 nucleotide 609 polymorphism in prostate carcinogenesis.     

Significant Overexpression of DVL1 in Taiwanese Colorectal Cancer Patients with Liver Metastasis

Undetected micrometastasis plays a key role in the metastasis of cancer in colorectal cancer (CRC) patients. The aim of this study is to identify a biomarker of CRC patients with liver metastasis through the detection of circulating tumor cells (CTCs). Microarray and bioinformatics analysis of 10 CRC cancer tissue specimens compared with normal adjacent tissues revealed that 31 genes were up-regulated (gene expression ratio of cancer tissue to paired normal tissue > 2) in the cancer patients. We used a weighted enzymatic chip array (WEnCA) including 31 prognosis-related genes to investigate CTCs in 214 postoperative stage I–III CRC patients and to analyze the correlation between gene expression and clinico-pathological parameters. We employed the immunohistochemistry (IHC) method with polyclonal mouse antibody against DVL1 to detect DVL1 expression in 60 CRC patients. CRC liver metastasis occurred in 19.16% (41/214) of the patients. Using univariate analysis and multivariate proportional hazards regression analysis, we found that DVL1 mRNA overexpression had a significant, independent predictive value for liver metastasis in CRC patients (OR: 5.764; 95% CI: 2.588–12.837; p < 0.0001 on univariate analysis; OR: 3.768; 95% CI: 1.469–9.665; p = 0.006 on multivariate analysis). IHC staining of the immunoreactivity of DVL1 showed that DVL1 was localized in the cytoplasm of CRC cells. High expression of DVL1 was observed in 55% (33/60) of CRC tumor specimens and was associated significantly with tumor depth, perineural invasion and liver metastasis status (all p < 0.05). Our experimental results demonstrated that DVL1 is significantly overexpressed in CRC patients with liver metastasis, leading us to conclude that DVL1 could be a potential prognostic and predictive marker for CRC patients.     

Pro-Apoptotic Effect of Rice Bran Inositol Hexaphosphate (IP6) on HT-29 Colorectal Cancer Cells

Inositol hexaphosphate (IP₆), or phytic acid is a natural dietary ingredient and has been described as a “natural cancer fighter”, being an essential component of nutritional diets. The marked anti-cancer effect of IP₆ has resulted in our quest for an understanding of its mechanism of action. In particular, our data provided strong evidence for the induction of apoptotic cell death, which may be attributable to the up-regulation of Bax and down-regulation of Bcl-xl in favor of apoptosis. In addition, the up-regulation of caspase-3 and -8 expression and activation of both caspases may also contribute to the apoptotic cell death of human colorectal adenocarcinoma HT-29 cells when exposed to IP₆. Collectively, this present study has shown that rice bran IP₆ induces apoptosis, by regulating the pro- and anti-apoptotic markers; Bax and Bcl-xl and via the activation of caspase molecules (caspase-3 and -8).     

Clinicopathologic and Prognostic Association of GRP94 Expression in Colorectal Cancer with Synchronous and Metachronous Metastases

Patients with advanced colorectal cancer (CRC) with distant metastases have a poor prognosis. We evaluated the clinicopathological relevance of GRP94 expression in these cases. The immunohistochemical expression of GRP94 was studied in 189 CRC patients with synchronous (SM; n = 123) and metachronous metastases (MM; n = 66), using tissue microarray; the association between GRP94 expression, outcome, and tumor-infiltrating lymphocytes (TILs) was also evaluated. GRP94 was expressed in 64.6% (122/189) patients with CRC; GRP94 positivity was found in 67.5% and 59.1% patients with SM and MM, respectively. In the SM group, high GRP94 expression was more common in patients with a higher density of CD4+ TILs (p = 0.002), unlike in the MM group. Survival analysis showed that patients with GRP94 positivity had significantly favorable survival (p = 0.030); after multivariate analysis, GRP94 only served as an independent prognostic factor (p = 0.034; hazard ratio, 0.581; 95% confidence interval, 0.351–0.961) in the SM group. GRP94 expression was detected in 49.4% of metastatic sites and showed significant heterogeneity between primary and metastatic lesions (p = 0.012). GRP94 is widely expressed in CRC with distant metastases; its expression was associated with favorable prognosis in the SM group, unlike in the MM group.     

Insulin-like Growth Factor Binding Protein-2 (IGFBP2) Is a Key Molecule in the MACC1-Mediated Platelet Communication and Metastasis of Colorectal Cancer Cells

Tumor cell crosstalk with platelets and, subsequently, their activation are key steps in hematogenous tumor metastasis. MACC1 is an oncogene involved in molecular pathogenesis of colorectal cancer (CRC) and other solid tumor entities, mediating motility and metastasis, making MACC1 an accepted prognostic biomarker. However, the impact of MACC1 on platelet activation has not yet been addressed. Here, we investigated the activation of platelets by human CRC cells upon MACC1 modulation, indicated by platelet aggregation and granule release. These approaches led to the identification of insulin-like growth factor binding protein-2 (IGFBP2) as a functional downstream molecule of MACC1, affecting communication with platelets. This was confirmed by an shRNA-mediated IGFBP2 knockdown, while maintaining MACC1 activity. Although IGFBP2 displayed an attenuated platelet activation potential, obviously by scavenging IGF-I as a platelet costimulatory mediator, the MACC1/IGFBP2 axis did not affect the thrombin formation potential of the cells. Furthermore, the IGFBP2/MACC1-driven cell migration and invasiveness was further accelerated by platelets. The key role of IGFBP2 for the metastatic spread in vivo was confirmed in a xenograft mouse model. Data provide evidence for IGFBP2 as a downstream functional component of MACC1-driven metastasis, linking these two accepted oncogenic biomarkers for the first time in a platelet context.     

Sequential Targeting of PLK1 and PARP1 Reverses the Resistance to PARP Inhibitors and Enhances Platin-Based Chemotherapy in BRCA-Deficient High-Grade Serous Ovarian Cancer with KRAS Amplification

Ovarian cancer (OC) accounts for approximately 4% of cancer deaths in women worldwide and is the deadliest gynecologic malignancy. High-grade serous ovarian cancer (HGSOC) is the most predominant ovarian cancer, in which BRCA1/2 gene mutation ranges from 3 to 27%. PARP inhibitors (PARPi) have shown promising results as a synthetically lethal therapeutic approach for BRCA mutant and recurrent OC in clinical use. However, emerging data indicate that BRCA-deficient cancers may be resistant to PARPi, and the mechanisms of this resistance remain elusive. We found that amplification of KRAS likely underlies PARPi resistance in BRCA2-deficient HGSOC. Our data suggest that PLK1 inhibition restores sensitivity to PARPi in HGSOC with KRAS amplification. The sequential combination of PLK1 inhibitor (PLK1i) and PARPi drastically reduces HGSOC cell survival and increases apoptosis. Furthermore, we were able to show that a sequential combination of PLK1i and PARPi enhanced the cellular apoptotic response to carboplatin-based chemotherapy in KRAS-amplified resistant HGSOC cells and 3D spheroids derived from recurrent ovarian cancer patients. Our results shed new light on the critical role of PLK1 in reversing PARPi resistance in KRAS-amplified HGSOC, and offer a new therapeutic strategy for this class of ovarian cancer patients where only limited options currently exist.     

p130Cas Is Correlated with EREG Expression and a Prognostic Factor Depending on Colorectal Cancer Stage and Localization Reducing FOLFIRI Efficacy

p130 Crk-associated substrate (p130Cas) is associated with poor prognosis and treatment resistance in breast and lung cancers. To elucidate p130Cas functional and clinical role in colorectal cancer (CRC) progression/therapy resistance, we performed cell culture experiments and bioinformatic/statistical analyses of clinical data sets. p130Cas expression was associated with poor survival in the cancer genome atlas (TCGA) data set. Knockdown/reconstitution experiments showed that p130Cas drives migration but, unexpectedly, inhibits proliferation in CRC cells. TCGA data analyses identified the growth factor epiregulin (EREG) as inversely correlated with p130Cas. p130Cas knockdown and simultaneous EREG treatment further enhanced proliferation. RNA interference and EREG treatment experiments suggested that p130Cas/EREG limit each other’s expression/activity. Inverse p130Cas/EREG Spearman correlations were prominent in right-sided and earlier stage CRC. p130Cas was inducible by 5-fluorouracil (5-FU) and FOLFIRI (folinic acid, 5-FU, irinotecan), and p130Cas and EREG were upregulated in distant metastases ({"type":"entrez-geo","attrs":{"text":"GSE121418","term_id":"121418"}}GSE121418). Positive p130Cas/EREG correlations were observed in metastases, preferentially in post-treatment samples (especially pulmonary metastases). p130Cas knockdown sensitized CRC cells to FOLFIRI independent of EREG treatment. RNA sequencing and gene ontology analyses revealed that p130Cas is involved in cytochrome P450 drug metabolism and epithelial-mesenchymal transition. p130Cas expression was associated with poor survival in right-sided, stage I/II, MSS (microsatellite stable), or BRAF-mutated CRC. In summary, p130Cas represents a prognostic factor and potential therapeutic target in CRC.     

The Multiple Cellular Roles of SMUG1 in Genome Maintenance and Cancer

Single-strand selective monofunctional uracil DNA glycosylase 1 (SMUG1) works to remove uracil and certain oxidized bases from DNA during base excision repair (BER). This review provides a historical characterization of SMUG1 and 5-hydroxymethyl-2′-deoxyuridine (5-hmdU) one important substrate of this enzyme. Biochemical and structural analyses provide remarkable insight into the mechanism of this glycosylase: SMUG1 has a unique helical wedge that influences damage recognition during repair. Rodent studies suggest that, while SMUG1 shares substrate specificity with another uracil glycosylase UNG2, loss of SMUG1 can have unique cellular phenotypes. This review highlights the multiple roles SMUG1 may play in preserving genome stability, and how the loss of SMUG1 activity may promote cancer. Finally, we discuss recent studies indicating SMUG1 has moonlighting functions beyond BER, playing a critical role in RNA processing including the RNA component of telomerase.     

No Association between HMOX1 and Risk of Colorectal Cancer and No Interaction with Diet and Lifestyle Factors in a Prospective Danish Case-Cohort Study

Red meat is a risk factor for colorectal cancer (CRC). We wanted to evaluate whether a functional polymorphism in the HMOX1 gene encoding heme oxygenase modifies risk of CRC or interacts with diet or lifestyle factors because this would identify heme or heme iron as a risk factor of CRC. The HMOX1 A-413T (rs2071746) was assessed in relation to risk of colorectal cancer (CRC) and interactions with diet (red meat, fish, fiber, cereals, fruit and vegetables) and lifestyle (use of non-steroidal anti-inflammatory drug and smoking status) were assessed in a case-cohort study of 928 CRC cases and a comparison group of 1726 randomly selected participants from a prospective study of 57,053 persons. No association between HMOX1 A-413T and CRC risk was found (TT vs. AA + TA; IRR = 1.15, 95% CI: 0.98–1.36, p = 0.10 for the adjusted estimate). No interactions were found between diet or lifestyle and HMOX1 A-413T. HMOX1 A-413T was not associated with CRC risk and no interactions with diet or lifestyle were identified in this large, prospective cohort with high meat intake. The results reproduced the previous findings from the same cohort and did not support a link between heme or heme iron and colorectal cancer. These results should be sought and replicated in other well-characterized cohorts with high meat intake.     

Relationships of Gut Microbiota Composition,Short-Chain Fatty Acids and Polyamines with the Pathological Response to Neoadjuvant Radiochemotherapy in Colorectal Cancer Patients

Emerging evidence has suggested that dysbiosis of the gut microbiota may influence the drug efficacy of colorectal cancer (CRC) patients during cancer treatment by modulating drug metabolism and the host immune response. Moreover, gut microbiota can produce metabolites that may influence tumor proliferation and therapy responsiveness. In this study we have investigated the potential contribution of the gut microbiota and microbial-derived metabolites such as short chain fatty acids and polyamines to neoadjuvant radiochemotherapy (RCT) outcome in CRC patients. First, we established a profile for healthy gut microbiota by comparing the microbial diversity and composition between CRC patients and healthy controls. Second, our metagenomic analysis revealed that the gut microbiota composition of CRC patients was relatively stable over treatment time with neoadjuvant RCT. Nevertheless, treated patients who achieved clinical benefits from RTC (responders, R) had significantly higher microbial diversity and richness compared to non-responder patients (NR). Importantly, the fecal microbiota of the R was enriched in butyrate-producing bacteria and had significantly higher levels of acetic, butyric, isobutyric, and hexanoic acids than NR. In addition, NR patients exhibited higher serum levels of spermine and acetyl polyamines (oncometabolites related to CRC) as well as zonulin (gut permeability marker), and their gut microbiota was abundant in pro-inflammatory species. Finally, we identified a baseline consortium of five bacterial species that could potentially predict CRC treatment outcome. Overall, our results suggest that the gut microbiota may have an important role in the response to cancer therapies in CRC patients.     

Inhibition of α(1,6)fucosyltransferase: Effects on Cell Proliferation,Migration, and Adhesion in an SW480/SW620 Syngeneic Colorectal Cancer Model

The present study explored the impact of inhibiting α(1,6)fucosylation (core fucosylation) on the functional phenotype of a cellular model of colorectal cancer (CRC) malignization formed by the syngeneic SW480 and SW620 CRC lines. Expression of the FUT8 gene encoding α(1,6)fucosyltransferase was inhibited in tumor line SW480 by a combination of shRNA-based antisense knockdown and Lens culinaris agglutinin (LCA) selection. LCA-resistant clones were subsequently assayed in vitro for proliferation, migration, and adhesion. The α(1,6)FT-inhibited SW480 cells showed enhanced proliferation in adherent conditions, unlike their α(1,6)FT-depleted SW620 counterparts, which displayed reduced proliferation. Under non-adherent conditions, α(1,6)FT-inhibited SW480 cells also showed greater growth capacity than their respective non-targeted control (NTC) cells. However, cell migration decreased in SW480 after FUT8 knockdown, while adhesion to EA.hy926 cells was significantly enhanced. The reported results indicate that the FUT8 knockdown strategy with subsequent selection for LCA-resistant clones was effective in greatly reducing α(1,6)FT expression in SW480 and SW620 CRC lines. In addition, α(1,6)FT impairment affected the proliferation, migration, and adhesion of α(1,6)FT-deficient clones SW480 and SW620 in a tumor stage-dependent manner, suggesting that core fucosylation has a dynamic role in the evolution of CRC.