Faraz Arshad Malik

Mutational spectrum of Gelsolin and its down regulation is associated with breast cancer.

Cytoskeletal rearrangement occurs in variety of cellular processes and involves a wide spectrum of proteins. Gelsolin super family proteins control actin organization by severing and capping filament ends and nucleating actin assembly. Gelsolin is the founding member of this family and plays important role in pathogenesis of human neoplasia. This study aimed to investigate the germline mutations and expressional profile of Gelsolin in human breast cancer tissues. For germ line screening PCR-SSCP technique was used while expression was analyzed through quantitative real time PCR. Different types of mutations were observed in Gelsolin coding regions on exons 4, 10, 11, 14 and 15. These mutations include 3 missense nonsynonymous substitution mutations, 2 deletions, 1 insertion and 1 synonymous substitution mutation. Gelsolin transcript level was found significantly lower in breast tumor tissues compared to control samples (p=0.03). Low level of Gelsolin was found in metastatic patients (p=0.002) and patients who died from breast cancer (P=0.03) compared to disease free patients at final follow up. This study shows that level of Gelsolin is down regulated in breast cancer tissues and is linked with metastasis development and death in patients. It is concluded that genetic changes in coding regions of Gelsolin can potentially contribute to genetic instability. These genetic variations and expressional correlation with patient survival may prove to be of significant importance.

Promoter Methylation Status Modulate the Expression of Tumor Suppressor (RbL2/p130) Gene in Breast Cancer

Background Aberrant expression of tumor suppressor genes may correspond to the abnormal cell development and tumorigenesis. Rbl2/p130, a member of retinoblastoma family of proteins, has growth suppressive properties. Numerous studies reported de-regulation of Rbl2/p130 in various types of cancer as a consequence of a number of genetic alterations. However, role of epigenetic mechanisms like DNA methylation in Rbl2/p130 expression remains elusive. Methods In the current study, 76 breast cancer tumors along with normal tissues (n = 76), blood (n = 76) of respective individuals and control blood (n = 50) were analyzed. Rbl2/p130 expression was analyzed by quantitative real time PCR (syber green method). Promoter methylation status was studied through methylation specific PCR of bisulfite converted genomic DNA. Data was analyzed using various statistical tests. Results We report significantly reduced Rbl2/p130 expression (P = 0.001) in tumors tissues as compared to control samples. Similarly, Rbl2/p130 expression varies with age and disease stages (P = 0.022), which suggest its involvement in tumor progression. Aberrant promoter methylation (Δmeth) was found in almost all the diseased samples and that was significantly different (P<0.001) with control samples. Similarly, methylation status varies significantly with tumor progression stages (P = 0.022). Hyper-methylation was observed at -1, +3, +15 and +75 of Rbl2/p130 promoter flanking around the TSS. Statistical analysis revealed that Rbl2/p130 expression negatively correlates to its promoter methylation (r = -0.412) in tumor tissues. Our results reflect an epigenetic regulation of Rbl2/p130 expression in breast cancer. This highlights the importance of Rbl2/p130 promoter methylation in breast cancer pathogenesis.

Unusual Intronic Variant in GSTP1 in Head and Neck Cancer in Pakistan

In the present case control study mRNA expression of the GSTP1 gene, encoding a phase II enzyme that detoxifies via glutathione conjugation, was investigated using semiquantitative PCR followed by SSCP for 49 confirmed head and neck (HN) cancer and 49 control samples. It was found that GSTP1 was upregulated in significantly higher number of cancers (OR 4.2, 95% CI 1.2- 15.3). Grade wise correlation was also observed with more up regulation in patients with more advanced grades of HN carcinomas. We also found that 5 patients showed variation in mRNA with a larger product size than expected. Sequencing revealed insertion of an intronic segment between the 6th and 7th exon of the GSTP1 gene. Germline screening was performed showing mobility shifts which suggested mutation at the DNA level resulting in intronic portion retention. This study is of prime importance for drug design and treatment selection to overcome increased resistance of HN cancers to drugs due to alteration in the GSTP1 gene.

Prognostic Significance of Altered Blood and Tissue Glutathione Levels in Head and Neck Squamous Cell Carcinoma Cases

Glutathione is a thiol compound that plays an important role in the antioxidant defense system of the cell and its deficiency leads to an increased susceptibility to oxidative stress and, thus, progression of many disease states including head and neck cancer. In the present study, alterations of glutathione levels were investigated in study cohort of 500 samples (cohort 1 containing 200 head and neck cancer blood samples along with 200 healthy controls and cohort II with 50 head and neck squamous cell carcinoma tissue samples along with 50 control tissues) by high performance liquid chromatography. The results indicated that mean blood glutathione levels were significantly reduced in head and neck cancer patients (p<0.001) compared to respective controls. In contrast, the levels of glutathione total (p<0.05) and glutathione reduced (p<0.05) were significantly elevated in head and neck squamous cell carcinoma tissues compared to the adjacent cancer-free control tissues. In addition to this, pearson correlation performed to correlate different tissue glutathione levels (GSH) with clinical/ pathological parameters demonstrated a significant negative correlation between pT-stage and GSH level (r=- 0.263**; p<0.01), C-stage and GSH level (r=-0.335**; p<0.01), grade and GSH (r=-0.329**; p<0.01) and grade versus redox index (r=-0.213**; p<0.01) in HNSCC tissues. Our study suggests that dysregulation of glutathione levels in head and neck cancer has the potential to predict metastasis, and may serve as a prognostic marker.

Rb1/105 gene alterations and head and neck carcinogenesis

Retinoblastoma gene (Rb1) is a tumor suppressor gene, which plays a pivotal role in cell cycle regulation, promoting G1/S arrest and growth restriction through inhibition of the E2F transcription factor. Abnormalities in the genes involved in cell cycle, including Rb1, have been reported in head and neck cancer (HNC) patients. Studies regarding Rb1 have been observed in different world populations but data is missing for Pakistani population. This study was aimed to analyze the genetic aberrations of Rb1 and their association with the development of HNC in Pakistani population. Genomic DNA was isolated from blood samples of 300 HNC patients and 270 controls. Salient coding region of gene was amplified by using Polymerase Chain Reaction (PCR). PCR conditions were optimized for each exon separately. Amplified products were analyzed for mutational screening using Single strand confirmation polymorphism (SSCP) technique followed by sequence analysis. Sequence analysis revealed five missense mutations g77082G>C, g77083G>A, g170220A>T, g170221G>C, g170228T>A, two frameshift mutations, two stop codon and two intronic substitutions in this study. The overall frequency of these mutations was 0.71. Frequency of nonsense mutations; Lys462stop (Novel) and Ser834stop (CM952105) were 0.15 and 0.14 respectively. We also report here novel missense mutations, frameshift mutation and a stop codon Lys462stop in HNC patients of Pakistani origin.This study suggests that the Rb1 germline mutations may contribute to genetic susceptibility for HNC. To our knowledge, this is the first report that Rb1 gene may be associated with risk of cancer in Pakistani population.