STAT3 paradoxically stimulates ß-catenin expression but inhibits ß-catenin function.

Wnt signalling and the signal transducer and activator of transcription 3 (STAT3) are oncogenic signalling pathways which are deregulated in colorectal cancer (CRC). Here we investigated the interaction of these two pathways. Firstly, we investigated biochemical interaction by inhibiting STAT3 and ß-catenin (through gene knock-down and dominant-negative TCF4 expression) in nine CRC cell lines. ß-catenin inhibition did not affect STAT3 levels, whereas STAT3 knock-down resulted in reduced ß-catenin mRNA and protein levels. The reduction in ß-catenin protein was not prevented by proteasome inhibition, and IL6-induced STAT3 activation resulted in increased ß-catenin mRNA. This suggests that STAT3 positively regulates ß-catenin (at a transcriptional level) and evaluation of 44 CRCs by immunostaining supported this by showing an association between nuclear STAT3 expression and nuclear ß-catenin (P = 0.022). We tested the functional interaction between STAT3 and Wnt signalling by knocking down STAT3 and ß-catenin individually and in combination. Knock-down of ß-catenin and STAT3 individually inhibited cell proliferation (P < 0. 001 for each) through G1 arrest. However, simultaneous knock-down of STAT3 and ß-catenin had a significantly weaker effect than knock-down of ß-catenin alone (P < 0.01). Knock-down of STAT3 and ß-catenin, individually and together, inhibited cell motility (P < 0.001) without evidence of interaction. We conclude that STAT3 regulates ß-catenin but ß-catenin does not regulate STAT3. The STAT3/ß-catenin interaction is complex but may reduce the proliferative activity of ß-catenin possibly by taking ß-catenin protein beyond the optimal level. This may indicate biological differences in tumours where both STAT3 and ß-catenin are activated compared to those where only one is activated.

Demographic and clinico-pathological characteristics of colorectal cancer in Kirkuk governorate, Iraq.

BACKGROUND: World-wide Colorectal cancer (CRC) is the third most common cancer with one million new cases a year. Historically, a higher incidence of this disease has been recorded among the elderly in the western countries, but it is increasing in developing countries and in younger age groups. AIM: This study aims to find whether CRC cancer is progressively affecting the younger age groups known as early onset (< 50 years). In addition, it describes the pathological characteristics of CRC in early onset CRC cases. METHOD: The study is retrospective cross-sectional. It was conducted over a period of five months from October 1st 2019 till 1st March 1st 2020. Data were drawn from patients with CRC from their medical records at Kirkuk Oncology Centre (KOC) and from the IRAQI National CANCER REGISTRY (INCR) over thirteen years period from 2006 to 2018. The basic data we obtained for each patient include sex, age, and stage, grade of the disease at diagnosis and mode of presentation. RESULTS: The Initial study population included 654 patients of both genders and all ages. CRC occurred in < 5.5/100,000 population per year which accounted for < 8% of total malignancies (2006-2018). The patients were divided into two groups; an early onset (< 50 years) group and a late onset CRC (⩾ 50 years) group. The final study population provided enough data for 238 patients for the years (2014-2018) with an age range of 20-91 and a mean of 54.4 years. The males were ∼54% while ∼46% were females. The age group under 50 years (early onset CRC) was ∼41% (no 98) while those who are 50 years and older (late onset) stood for 59% (no 140). There were no statistical differences between the two age groups regarding stage, grade, or presenting symptom. CONCLUSION: CRC is common in early onsets or young age groups with similar pathological characteristics to those of the late onset cancer. Accordingly, even mild lower gastrointestinal symptoms should be taken seriously. The study points toward an increasing awareness of the population on the importance of colorectal cancer. Also, conducting more surveillance studies and investigations would be recommended for early detections of the disease in young populations.

The utility of diagnostic biopsy specimens for predictive molecular testing in colorectal cancer.

AIMS: If stratified medicine is to be applied in the neoadjuvant setting, predictive testing will have to be undertaken on preoperative diagnostic biopsy specimens. The aim of this study was to evaluate whether a diagnostic biopsy was adequately representative of the main tumour in colorectal cancer. METHODS AND RESULTS: Thirty cases of paired biopsy and subsequent resection specimens were randomly selected. Samples were screened for mutation in KRAS (codons 12/13, 61, and 146), BRAF (codon 600 and exon 11), PIK3CA (exons 1, 9, and 20), TP53 (exons 5-8), and microsatellite instability, using the quick multiplex consensus or standard polymerase chain reaction (PCR) protocols followed by high-resolution melting analysis. A total of 570 paired PCR tests were performed for mutation detection, and identical results were obtained in both biopsy and resection specimens in 569 tests (>99% concordance). Four cases (13%) showed microsatellite instability, and, in all four cases, instability was seen at identical mononucleotide markers in both biopsy and matched resection specimens. CONCLUSIONS: This is the first study to show that diagnostic biopsy specimens, even though they are a tiny sample of the tumour, are sufficiently representative for use in predictive testing for early driver mutations in colorectal cancer.

Trends in colorectal cancer in Iraq over two decades: incidence, mortality, topography and morphology.

BACKGROUND: Colorectal cancer (CRC) is mainly a disease of the elderly in the Western world, but its characteristics are changing globally. Iraq does not have a well established CRC screening program. Understanding trends of CRC incidence, fatality and the clinical features of CRC patients is vital to the design of effective public health measures; public awareness, screening, diagnosis and treatment strategies to meet the future demands. OBJECTIVES: Determine trends in demography, incidence proportion, mortality, topography (primary tumor site) and morphology (histology) over two decades. DESIGN: Registry-based study SETTING: Iraqi National Cancer Registry (INCR) database PATIENTS AND METHODS: We collected and analyzed data from CRC patients obtained from the INCR to calculate incidence and mortality proportion per 100 000 population for the period from 2000 to 2019. In addition to estimation, data were examined by anatomic location and morphological type. MAIN OUTCOME MEASURES: Change in the incidence and mortality proportion, topography and morphology of CRC over 20 years. SAMPLE SIZE: 20 880 CRC patients ranging in age from 14-80 years. RESULTS: The overall (males and females) CRC incidence proportion (CIP) increased from 2.28 to 6.18 per 100 000 population in 2000 and 2019, respectively, with an annual percentage change (APC) of 5.11%. The incidence proportion (IP) of CRC in patients from 20 to <50 years rose from 1.46 in 2000 to 4.36 per 100 000 population in 2019, which is an APC of 5.6%. The IP in patients older than 50 years rose from 12.7 to 40.59 per 100 000 population in 2000 and 2019, respectively, with an APC of 5.98%. The percentage of all CRC cases to all total malignancies in Iraq grew from 3.69% in 2000 to 6.5% in 2019. The CRC mortality proportion increased from 1.25 to 1.77 per 100 000 populations in 2010 and 2019, respectively, reflecting an APC of 3.54%. Anatomically, colon (C18) tumor represented 59.2% and 65.7% in 2000 and 2019, respectively. Rectal (C20) tumors were 37.2% in 2000 down to 31.4% in 2019, while rectosigmoid junction tumor (C19) were 3.6% in 2000 dropping to 2% in 2019. CONCLUSIONS: CRC in Iraq is still a disease of the elderly and is rising in incidence and mortality in all age groups. This necessitates reconsidering health policy regarding CRC; public awareness, screening and management strategies to accommodate for these alarming changes. LIMITATIONS: Data about stages, grades and molecular characterisations are not available in the INCR. CONFLICT OF INTEREST: None.

Genetic Variations in Cytochrome P450 1A1 and 1B1 Genes in a Cohort of Patients from Iraq Diagnosed with Breast Cancer.

Breast cancer is the most prevalent malignant neoplasm in females. Genetic variations in the xenobiotic metabolising cytochrome enzymes; Family 1 Subfamily A Member 1 (CYP1A1) and Family 1 Subfamily B Member 1 (CYP1B1) might play a role in the individual susceptibility to breast cancer and its prognosis. The goal of this study is to estimate the incidence of single nucleotide polymorphisms (SNPs) in CYP1A1 (rs1048943, Ile462VaI, and rs4646903/MSP1) and in CYP1B1 (rs1056836, Leu432Val) genes in patients with breast cancer. This case-control study included 180 female patients with breast cancer and 180 healthy control subjects from Kirkuk/Iraq. Genomic DNA was extracted from venous blood samples and tested for SNPs by the direct DNA sequencing technique. A statistical analysis was done to identify if there is any association between SNPs and the increasing odd of breast cancer and its stage, grade and molecular subtype at diagnosis. The common (reference) genotype of CYP1A1 gene rs1048943 is AA. The AG and GG variant genotypes were significantly more common in the breast cancer patients and conferred an increased odd of breast cancer and its later stages (stages III and IV) and poor differentiation (P < .01) but not with the molecular subtypes. The common genotype of CYP1A1 rs4646903 is TT. The variant genotypes TC and CC are not associated either with increased risk of breast cancer (P > .05) or with its stage, grade or molecular subtypes (P > .05). The GG genotype of CYP1B1 rs1056836 was the common genotype. The CG and CC variant genotypes were not associated with the increased risks of breast cancer (P > .05) or its stage, grade or molecular subtypes (P > .05). In conclusion, variants genotypes of CYP1A1 rs1048943 might play a role in breast cancer pathogenesis and prognosis and can have a place in cancer screening and tailored medicine in the future in the Iraqi population. Future larger scale studies including other genes might help to better understand the role of the SNP in breast risk and its prognosis.

Sequence Polymorphism in Xenobiotic Metabolising Genes in Iraqi Colorectal Cancer Patients.

OBJECTIVES: Colorectal cancer (CRC) is the third most prevalent malignant neoplasm. Genetic variations in the xenobiotic metabolising cytochrome enzymes. Family 1 Subfamily A Member 1 (CYP1A1) and Family 1 Subfamily B Member 1 (CYP1B1) might play a role in cancer pathogenesis and prognosis. The aim of this work is to determine the frequency of Single Nucleotide Polymorphisms (SNPs) in CYP1A1 (rs1048943, Ile462VaI and rs4646903/MSP1) and CYP1B1 (rs1056836, Leu432Val) genes in patients with CRC cancer. It was also an attempt to identify the association between SNPs and CRC and its stage and grade at diagnosis. METHODS: This case-control study was conducted in Kirkuk/Iraq, 200 patients with CRC and 200 cancer free control subjects were enrolled. Genomic DNA was extracted from venous blood samples and screened for SNPs using Restriction Fragment Length  Polymorphism (RFLP) and confirmed by the direct DNA sequencing. RESULTS: The reference genotype of CYP1A1 gene rs1048943 is AA. Both the AG and GG variants were  significantly more frequent in the cancer group and associated with increased risks of CRC and its later stages (stages III and IV)  and poor  differentiation (p <0.01). The reference genotype of CYP1A1 rs4646903 is TT. The variant genotypes, TC and CC, had no significant association with increased odds of cancer (P>0.05) or with tumour stage or its grade (p>0.05). The GG genotype of CYP1B1 rs1056836 was the reference genotype. The CG and CC variants were not associated with increased risks of CRC (P>0.05) or its stage or grade except the CG genotype which was associated with poor differentiation (OR= 3.4, 95 % CI= 1.8 -6.5, p <0.001). CONCLUSION: CYP1A1 gene rs1048943 SNPs can represent a potential future marker for   CRC risk prediction and prognosis. Further evaluation in large scale studies will provide greater understanding of the effects of other genes SNPs on CRC  risk and prognosis.
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Comparative analysis of pyrosequencing and QMC-PCR in conjunction with high resolution melting for KRAS/BRAF mutation detection.

Mutation detection is important in cancer management. Several methods are available of which high resolution melting (HRM) analysis and pyrosequencing are the most versatile. We undertook a comparative analysis of these techniques. The methods are: To compare the limit of detection (LOD), mutations in KRAS (codon 12/13 hotspot) and BRAF (V600E hotspot) were tested. DNA mixtures containing mutant alleles at a frequency of around 25%/12.5%/6%/3%/ 1.5%/0.8% were analysed. To compare frequency of mutation detection, 22 DNA samples (nine high quality samples from cell lines, 13 low quality samples from formalin-fixed paraffin-embedded tissue) were tested for three hotspots in KRAS (codons 12/13, 61 and 146) and two hotspots in BRAF (V600E and exon 11). HRM analysis of KRAS (codon12/13) and BRAF (V600E) showed that 3% and 1.5% mutant alleles respectively could be reliably detected whilst pyrosequencing reliably detected 6% mutant alleles in each case. Of 110 tests performed on 22 DNA samples, in 109 cases HRM and pyrosequencing gave identical results. Two of the samples tested had previously been called as wild type for KRAS by direct Sanger sequencing but were found to be mutant by both HRM and pyrosequencing. Both HRM and pyrosequencing can detect small numbers of mutant alleles although HRM has a lower limit of detection. Both are suitable for use in mutation detection and are both more sensitive than Sanger sequencing.

Genotoxic Effect of Lead and Cadmium on Workers at Wastewater Plant in Iraq.

Heavy metal poisoning is a worldwide problem that is caused by different human industrial activities such as battery and painting manufacturing and occupational exposure of those working at petrol stations. Wastewater is known to contain higher amounts of heavy metals such as lead (Pd) and cadmium (Cd) and might be sources of exposure for workers at the wastewater treatment plant. However, to our best knowledge, no studies were done to evaluate the level of cadmium and lead in blood of workers at wastewater treatment plants and evaluate the subsequent effect of lead and cadmium on delta-aminolevulinic acid dehydratase (δ-ALAD), urinary delta-aminolevulinic acid (Uδ-ALA), and 8-hydroxy-2'-deoxyguanosine (8-OHdG) as markers of lead and cadmium toxicity. In this case-control study, 79 workers at the Al-Rustumiya wastewater plant in Baghdad, Iraq, and 40 control subjects were included. The levels of lead and cadmium were measured in blood of the study subjects using the atomic absorption spectroscopy (AAS) method. 8-OHdG was analysed using enzyme-linked immunosorbent assay (ELISA) technique. δ-ALAD and Uδ-ALA were estimated using spectrophotometry-based methods. Our work showed that workers had a significantly higher level of lead and cadmium when compared with the control group (P < 0.05), yet, still within the World Health Organization permissible limit. The level of both metals was positively associated with duration of work at the plant (P < 0.01). The activity of δ-ALAD was inversely associated with the lead level, while both Uδ-ALA and 8-OHdG were positively correlated with the lead level (P < 0.05). These three markers lacked any statistically significant association with the cadmium level (P > 0.05). To sum up, working at the wastewater treatment plant was associated with a higher blood level of lead and cadmium and their possible health hazard. Health and occupational safety authorities are required to set up tighter regulations and protocols to minimize these hazards and ensure a safe working environment.

Cten is targeted by Kras signalling to regulate cell motility in the colon and pancreas.

CTEN/TNS4 is an oncogene in colorectal cancer (CRC) which enhances cell motility although the mechanism of Cten regulation is unknown. We found an association between high Cten expression and KRAS/BRAF mutation in a series of CRC cell lines (p = 0.03) and hypothesised that Kras may regulate Cten. To test this, Kras was knocked-down (using small interfering (si)RNA) in CRC cell lines SW620 and DLD1 (high Cten expressors and mutant for KRAS). In each cell line, Kras knockdown was mirrored by down-regulation of Cten Since Kras signals through Braf, we tested the effect of Kras knockdown in CRC cell line Colo205 (which shows high Cten expression and is mutant for BRAF but wild type for KRAS). Cten levels were unaffected by Kras knockdown whilst Braf knockdown resulted in reduced Cten expression suggesting that Kras signals via Braf to regulate Cten. Quantification of Cten mRNA and protein analysis following proteasome inhibition suggested that regulation was of Cten transcription. Kras knockdown inhibited cell motility. To test whether this could be mediated through Cten, SW620 cells were co-transfected with Kras specific siRNAs and a Cten expression vector. Restoring Cten expression was able to restore cell motility despite Kras knockdown (transwell migration and wounding assay, p<0.001 for both). Since KRAS is mutated in many cancers, we investigated whether this relationship could be demonstrated in other tumour models. The experiments were repeated in the pancreatic cancer cell lines Colo357 & PSN-1(both high Cten expressors and mutant for KRAS). In both cell lines, Kras was shown to regulate Cten and forced expression of Cten was able to rescue loss of cell motility following Kras knockdown in PSN-1 (transwell migration assay, p<0.001). We conclude that, in the colon and pancreas, Cten is a downstream target of Kras and may be a mechanism through which Kras regulates of cell motility.

Quick-multiplex-consensus (QMC)-PCR followed by high-resolution melting: a simple and robust method for mutation detection in formalin-fixed paraffin-embedded tissue.

BACKGROUND: Mutation detection in tumours will become increasingly important in pathological diagnosis as 'predictive' mutations are identified. A cheap and reliable test that works on formalin-fixed paraffin-embedded (FFPE) tissue is required. METHODS: The quick-multiplex-consensus (QMC)-PCR protocol was developed to be used with high-resolution melting (HRM) analysis. The assay was compared with Sanger sequencing. Robustness of the assay was tested in DNA from FFPE tissue. RESULTS: QMC-PCR with HRM could detect a minimum of 2.5% of mutant alleles (compared with 20% detectable for Sanger sequencing). Ten mutation hotspots in KRAS, BRAF, PIK3CA and CDC4 were screened in 29 cell lines with 100% sensitivity and specificity. Forty-three FFPE colorectal tumours were sequenced for hotspots in KRAS and PIK3CA and then screened by QMC-PCR. There was 100% sensitivity, although, of 21 mutations detected by QMC-PCR, 16 were confirmed by sequencing (71% specificity, positive predictive value 76%). All 43 samples were then screened for mutations in all 10 hotspots. Of 430 tests, 43 (10%) showed aberrant melting and 36 were confirmed mutant (positive predictive value 84%). As our technique is more sensitive than direct sequencing, the remaining seven tests are probably sequencing false-negatives. Precision tests showed that there was little intra-assay and interassay variation. CONCLUSIONS: QMC-PCR with HRM is a simple, robust and inexpensive technique which had greater sensitivity than Sanger sequencing. It allows multiple mutation hotspots to be rapidly screened and is thus highly suited to mutation detection in DNA derived from FFPE tissues.