GLI Transcriptional Targets S100A7 and KRT16 Show Upregulated Expression Patterns in Epidermis Overlying the Tumor Mass in Melanoma Samples.

Although not completely understood, the role of the Hedgehog-GLI (HH-GLI) signaling pathway in melanoma and epithelial skin tumors has been reported before. In this study, we confirmed in various melanoma cell line models that keratin 16 (KRT16) and S100 Calcium-Binding Protein A7 (S100A7) are transcriptional targets of GLI Family Zinc Finger (GLI) proteins. Besides their important role in protecting and maintaining the epidermal barrier, keratins are somehow tightly connected with the S100 family of proteins. We found that stronger expression of KRT16 indeed corresponds to stronger expression of S100A7 in our clinical melanoma samples. We also report a trend regarding staining of GLI1, which corresponds to stronger staining of GLI3, KRT16, and S100A7 proteins. The most interesting of our findings is that all the proteins are detected specifically in the epidermis overlying the tumor, but rarely in the tumor itself. The examined proteins were also not detected in the healthy epidermis at the edges of the sample, suggesting that the staining is specific to the epidermis overlaying the tumor mass. Of all proteins, only S100A7 demonstrated a statistically significant trend regarding tumor staging and staining intensity. Results from our clinical samples prove that immune infiltration is an important feature of melanoma. Pigmentophages and tumor-infiltrating lymphocytes (TIL) demonstrate a significant association with tumor stage, while mononuclear cells are equally present in all stages. For S100A7, we found an association between the number of TILs and staining intensity. Considering these new findings presented in our study, we suggest a more detailed examination of the possible role of the S100A7 protein as a biomarker in melanoma.

BIRC5 Gene Polymorphisms Are Associated with a Higher Stage of Local and Regional Disease in Oral and Oropharyngeal Squamous Cell Carcinomas.

Oral squamous cell carcinoma (OSCC) and oropharyngeal squamous cell carcinoma (OPSCC) are the most common types of cancers in the head and neck region (HNSCC). Despite very aggressive treatment modalities, the five-year survival rate has not changed for decades and is still around 60%. The search for potential specific biomarkers of aggressiveness or outcome indicators could be of great benefit in improving the treatment of these patients. One of the potential biomarkers is survivin, the protein product of the BIRC5 gene. In this study, we investigated the occurrence of BIRC5 gene polymorphisms in 48 patients with OSCC and OPSCC compared with healthy controls. A total of 18 polymorphisms were found, 11 of which occurred in HNSCC with a minor allele frequency (MAF) of more than 5%. Five polymorphisms (rs3764383, rs9904341, rs2071214, rs2239680, rs2661694) were significantly associated with tumor size, tumor stage, and advanced regional disease, but had no impact on survival.

AQP3-Dependent PI3K/Akt Modulation in Breast Cancer Cells.

Aquaporin 3 (AQP3) is a peroxiporin, a membrane protein that channels hydrogen peroxide in addition to water and glycerol. AQP3 expression also correlates with tumor progression and malignancy and is, therefore, a potential target in breast cancer therapy. In addition, epithelial growth factor receptor (EGFR) plays an important role in breast cancer. Therefore, we investigated whether disruption of the lipid raft harboring EGFR could affect AQP3 expression, and conversely, whether AQP3 silencing would affect the EGFR/phosphoinositide-3-kinase (PI3K)/Protein kinase B (PKB or Akt) signaling pathway in breast cancer cell lines with different malignant capacities. We evaluated H2O2 uptake, cell migratory capacity, and expression of PI3K, pAkt/Akt in three breast cancer cell lines, MCF7, SkBr3, and SUM159PT, and in the nontumorigenic breast epithelial cell line MCF10A. Our results show different responses between the tested cell lines, especially when compared to the nontumorigenic cell line. Neither lipid raft disruption nor EGF stimuli had an effect on PI3K/Akt pathway in MCF10A cell line. AQP3-silencing in SkBr3 and SUM159PT showed that AQP3 can modulate PI3K/Akt activation in these cells. Interestingly, SUM159PT cells increase nuclear factor-E2-related factor 2 (NRF2) in response to lipid raft disruption and EGF stimuli, suggesting an oxidative-dependent response to these treatments. These results suggest that in breast cancer cell lines, AQP3 is not directly related to PI3K/Akt pathway but rather in a cell-line-dependent manner.

GANT61 and Lithium Chloride Inhibit the Growth of Head and Neck Cancer Cell Lines Through the Regulation of GLI3 Processing by GSK3ß.

Several signaling pathways are aberrantly activated in head and neck squamous cell carcinoma (HNSCC), including the Hedgehog-Gli (HH-GLI), WNT, EGFR, and NOTCH pathways. The HH-GLI pathway has mostly been investigated in the context of canonical signal transduction and the inhibition of the membrane components of the pathway. In this work we investigated the role of downstream inhibitors GANT61 and lithium chloride (LiCl) on cell viability, wound closure, and colony forming ability of HNSCC cell lines. Five HNSCC cell lines were treated with HH-GLI pathway inhibitors affecting different levels of signal transduction. GANT61 and LiCl reduce the proliferation and colony formation capabilities of HNSCC cell lines, and LiCl has an additional effect on wound closure. The major effector of the HH-GLI signaling pathway in HNSCC is the GLI3 protein, which is expressed in its full-length form and is functionally regulated by GSK3ß. LiCl treatment increases the inhibitory Ser9 phosphorylation of the GSK3ß protein, leading to increased processing of GLI3 from full-length to repressor form, thus inhibiting HH-GLI pathway activity. Therefore, downstream inhibition of HH-GLI signaling may be a promising therapeutic strategy for HNSCC.

Potential hot spot for de novo mutations in PTCH1 gene in Gorlin syndrome patients: a case report of twins from Croatia.

We describe a case of twins with sporadic Gorlin syndrome. Both twins had common Gorlin syndrome features including calcification of the falx cerebri, multiple jaw keratocysts, and multiple basal cell carcinomas, but with different expressivity. One brother also had benign testicular mesothelioma. We propose this tumor type as a possible new feature of Gorlin syndrome. Gorlin syndrome is a rare autosomal dominant disorder characterized by both developmental abnormalities and cancer predisposition, with variable expression of various developmental abnormalities and different types of tumors. The syndrome is primarily caused by mutations in the Patched 1 (PTCH1) gene, although rare mutations of Patched 2 (PTCH2) or Suppressor of Fused (SUFU) genes have also been found. Neither founder mutations nor hot spot locations have been described for PTCH1 in Gorlin syndrome patients. Although de novo mutations of the PTCH1 gene occur in almost 50% of Gorlin syndrome cases, there are a few recurrent mutations. Our twin patients were carriers of a de novo mutation in the PTCH1 gene, c.3364_3365delAT (p.Met1122ValfsX22). This is, to our knowledge, the first Gorlin syndrome-causing mutation that has been reported four independent times in distant geographical locations. Therefore, we propose the location of the described mutation as a potential hot spot for mutations in PTCH1.

Role of GLI Transcription Factors in Pathogenesis and Their Potential as New Therapeutic Targets.

GLI transcription factors have important roles in intracellular signaling cascade, acting as the main mediators of the HH-GLI signaling pathway. This is one of the major developmental pathways, regulated both canonically and non-canonically. Deregulation of the pathway during development leads to a number of developmental malformations, depending on the deregulated pathway component. The HH-GLI pathway is mostly inactive in the adult organism but retains its function in stem cells. Aberrant activation in adult cells leads to carcinogenesis through overactivation of several tightly regulated cellular processes such as proliferation, angiogenesis, EMT. Targeting GLI transcription factors has recently become a major focus of potential therapeutic protocols.

GSK3ß and Gli3 play a role in activation of Hedgehog-Gli pathway in human colon cancer - Targeting GSK3ß downregulates the signaling pathway and reduces cell proliferation.

The role of Hedgehog-Gli (Hh-Gli) signaling in colon cancer tumorigenesis has not yet been completely elucidated. Here we provide strong evidence of Hh-Gli signaling involvement in survival of colon cancer cells, with the main trigger of activation being deregulated GSK3ß. Our clinical data reveals high expression levels of GSK3ß and Gli3 in human colon cancer tissue samples, with positive correlation between GSK3ß expression and DUKES' stage. Further experiments on colon cancer cell lines have shown that a deregulated GSK3ß upregulates Hh-Gli signaling and positively affects colon cancer cell survival. We show that inhibition of GSK3ß with lithium chloride enhances Gli3 processing into its repressor form, consequently downregulating Hh-Gli signaling, reducing cell proliferation and inducing cell death. Analysis of the molecular mechanisms revealed that lithium chloride enhances Gli3-SuFu-GSK3ß complex formation leading to more efficient Gli3 cleavage and Hh-Gli signaling downregulation. This work proposes that activation of the Hh-Gli signaling pathway in colon cancer cells occurs non-canonically via deregulated GSK3ß. Gli3 seems to be the main pathway effector, highlighting the activator potential of this transcription factor, which is highly dependent on GSK3ß function and fine tuning of the Gli3-SuFu-GSK3ß platform.

Regulation of human PTCH1b expression by different 5' untranslated region cis-regulatory elements.

PTCH1 gene codes for a 12-pass transmembrane receptor with a negative regulatory role in the Hedgehog-Gli signaling pathway. PTCH1 germline mutations cause Gorlin syndrome, a disorder characterized by developmental abnormalities and tumor susceptibility. The autosomal dominant inheritance, and the evidence for PTCH1 haploinsufficiency, suggests that fine-tuning systems of protein patched homolog 1 (PTC1) levels exist to properly regulate the pathway. Given the role of 5' untranslated region (5'UTR) in protein expression, our aim was to thoroughly explore cis-regulatory elements in the 5'UTR of PTCH1 transcript 1b. The (CGG)n polymorphism was the main potential regulatory element studied so far but with inconsistent results and no clear association between repeat number and disease risk. Using luciferase reporter constructs in human cell lines here we show that the number of CGG repeats has no strong impact on gene expression, both at mRNA and protein levels. We observed variability in the length of 5'UTR and changes in abundance of the associated transcripts after pathway activation. We show that upstream AUG codons (uAUGs) present only in longer 5'UTRs could negatively regulate the amount of PTC1 isoform L (PTC1-L). The existence of an internal ribosome entry site (IRES) observed using different approaches and mapped in the region comprising the CGG repeats, would counteract the effect of the uAUGs and enable synthesis of PTC1-L under stressful conditions, such as during hypoxia. Higher relative translation efficiency of PTCH1b mRNA in HEK 293T cultured hypoxia was observed by polysomal profiling and Western blot analyses. All our results point to an exceptionally complex and so far unexplored role of 5'UTR PTCH1b cis-element features in the regulation of the Hedgehog-Gli signaling pathway.

Signaling Switching from Hedgehog-GLI to MAPK Signaling Potentially Serves as a Compensatory Mechanism in Melanoma Cell Lines Resistant to GANT-61.

BACKGROUND: Melanoma represents the deadliest skin cancer due to its cell plasticity which results in high metastatic potential and chemoresistance. Melanomas frequently develop resistance to targeted therapy; therefore, new combination therapy strategies are required. Non-canonical signaling interactions between HH-GLI and RAS/RAF/ERK signaling were identified as one of the drivers of melanoma pathogenesis. Therefore, we decided to investigate the importance of these non-canonical interactions in chemoresistance, and examine the potential for HH-GLI and RAS/RAF/ERK combined therapy. METHODS: We established two melanoma cell lines resistant to the GLI inhibitor, GANT-61, and characterized their response to other HH-GLI and RAS/RAF/ERK inhibitors. RESULTS: We successfully established two melanoma cell lines resistant to GANT-61. Both cell lines showed HH-GLI signaling downregulation and increased invasive cell properties like migration potential, colony forming capacity, and EMT. However, they differed in MAPK signaling activity, cell cycle regulation, and primary cilia formation, suggesting different potential mechanisms responsible for resistance occurrence. CONCLUSIONS: Our study provides the first ever insights into cell lines resistant to GANT-61 and shows potential mechanisms connected to HH-GLI and MAPK signaling which may represent new hot spots for noncanonical signaling interactions.

A low-grade ovarian carcinoma case with coincident LOH of PTCH1 and BRCA1, and a mutation in BRCA1.

We report a case of a 53-year-old woman with Grade 1 serous cystadenocarcinoma on her left ovary and metastatic serous adenocarcinoma on her right ovary. Serous carcinoma is the most common type of ovarian cancer, representing approximately half of all cases. Because of positive family history, the patient was referred for BRCA1/2 screening. Germline BRCA1 mutation c.676delT (p.C226VfsX8) was found, and in tumor tissue the normal allele was lost. Tumor tissue also had loss of heterozygosity in the PTCH1 gene, one of the major members of the Hedgehog-Gli (Hh-Gli) pathway. Gene expression analysis showed upregulation of the Hh-Gli pathway in both ovaries compared with healthy ovarian tissue. Primary cell culture was developed from the patient's tissue and showed downregulation of gene expression in response to cyclopamine, a Hh-Gli pathway inhibitor. The Hh-Gli signaling pathway may play a role in malignant transformation and metastasis of ovarian cancer.

RNA-seq and ChIP-seq Identification of Unique and Overlapping Targets of GLI Transcription Factors in Melanoma Cell Lines.

BACKGROUND: Despite significant progress in therapy, melanoma still has a rising incidence worldwide, and novel treatment strategies are needed. Recently, researchers have recognized the involvement of the Hedgehog-GLI (HH-GLI) signaling pathway in melanoma and its consistent crosstalk with the MAPK pathway. In order to further investigate the link between the two pathways and to find new target genes that could be considered for combination therapy, we set out to find transcriptional targets of all three GLI proteins in melanoma. METHODS: We performed RNA sequencing on three melanoma cell lines (CHL-1, A375, and MEL224) with overexpressed GLI1, GLI2, and GLI3 and combined them with the results of ChIP-sequencing on endogenous GLI1, GLI2, and GLI3 proteins. After combining these results, 21 targets were selected for validation by qPCR. RESULTS: RNA-seq revealed a total of 808 differentially expressed genes (DEGs) for GLI1, 941 DEGs for GLI2, and 58 DEGs for GLI3. ChIP-seq identified 527 genes that contained GLI1 binding sites in their promoters, 1103 for GLI2 and 553 for GLI3. A total of 15 of these targets were validated in the tested cell lines, 6 of which were detected by both RNA-seq and ChIP-seq. CONCLUSIONS: Our study provides insight into the unique and overlapping transcriptional output of the GLI proteins in melanoma. We suggest that our findings could provide new potential targets to consider while designing melanoma-targeted therapy.

SHH-N non-canonically sustains androgen receptor activity in androgen-independent prostate cancer cells.

Prostate cancer is the second most frequent cancer diagnosed in men worldwide. Localized disease can be successfully treated, but advanced cases are more problematic. After initial effectiveness of androgen deprivation therapy, resistance quickly occurs. Therefore, we aimed to investigate the role of Hedgehog-GLI (HH-GLI) signaling in sustaining androgen-independent growth of prostate cancer cells. We found various modes of HH-GLI signaling activation in prostate cancer cells depending on androgen availability. When androgen was not deprived, we found evidence of non-canonical SMO signaling through the SRC kinase. After short-term androgen deprivation canonical HH-GLI signaling was activated, but we found little evidence of canonical HH-GLI signaling activity in androgen-independent prostate cancer cells. We show that in androgen-independent cells the pathway ligand, SHH-N, non-canonically binds to the androgen receptor through its cholesterol modification. Inhibition of this interaction leads to androgen receptor signaling downregulation. This implies that SHH-N activates the androgen receptor and sustains androgen-independence. Targeting this interaction might prove to be a valuable strategy for advanced prostate cancer treatment. Also, other non-canonical aspects of this signaling pathway should be investigated in more detail and considered when developing potential therapies.

Report on mutation in exon 15 of the APC gene in a case of brain metastasis.

The study analyzes exon 15 of the adenomatous polyposis coli gene (APC) in a 49-year-old male patient with brain metastasis. The primary site was lung carcinoma. PCR method and direct DNA sequencing of the metastasis and autologous lymphocyte samples identified the presence of a somatic mutation. The substitution was at position 5883 G-A in the metastasis tissue. The mutation was confirmed by RFLP analysis using Msp I endonuclease, since the mutation strikes the Msp I restriction site. Immunohistochemical analysis revealed the lack of protein expression of this tumor suppressor gene. The main molecular activator of the wnt pathway, beta-catenin, was expressed, and located in the nucleus. The mutation is a silent mutation that might have consequences in the creation of a new splice site. Different single-base substitutions in APC exons need not only be evaluated by the predicted change in amino acid sequence, but rather at the nucleotide level itself. In our opinion, such silent mutations should also be incorporated in mutation detection rate and validation.

Involvement of p16 and PTCH in pathogenesis of melanoma and basal cell carcinoma.

The involvement of two tumor suppressors p16 and Ptch in pathogenesis of cutaneous melanomas and basal cell carcinomas (BCCs) was studied through expression of Ptch and p16 and genetic alterations in 9p21 region (p16) and in 9q22.3 region (PTCH) of chromosome 9. Immunohistochemical analyses of paraffin-embedded tissues with Ptch and p16 antibodies, typing for 9q22-q31 and 9p21 region with polymorphic markers and p16 and Ptch mutation detection was done. Higher expression of p16 and Ptch in melanoma and BCC of the skin was frequently detected in studied cases. However, allelic loss of PTCH region occurs more frequently in BCCs than loss of heterozygosity of p16 region. Both types of tumors, BCCs and melanomas, suggest involvement of Hh-Gli signaling pathway, but using different mechanisms.

Gorlin syndrome patient with large deletion in 9q22.32-q22.33 detected by quantitative multiplex fluorescent PCR.

BACKGROUND: Gorlin syndrome is a rare autosomal-dominant disorder characterized by a wide range of developmental abnormalities and various tumors. The syndrome is caused by mutations in PTCH1, a tumor suppressor gene located at 9q22.32. We describe a Gorlin syndrome case with typical features of the syndrome and no mutations in PTCH1, but with a large deletion of the 9q22 region that has rarely been described. OBJECTIVE: To fully characterize the large deletion in the patient. METHODS: In order to map the size and position of the deletion, we developed quantitative multiplex fluorescent PCR with polymorphic markers surrounding the PTCH1 gene, followed by long-range PCR and sequencing. RESULTS: The deleted segment of 4.5 Mb in the 9q22.32-q22.33 region was determined, and included the entire PTCH1, its promoter and 22 OMIM genes. CONCLUSION: We suggest that screening for large deletions should be included in standard mutation screening for Gorlin syndrome patients.

Expression profiles of p53/p73, NME and GLI families in metastatic melanoma tissue and cell lines.

Unlike other tumours, TP53 is rarely mutated in melanoma; however, it fails to function as a tumour suppressor. We assume that its functions might be altered through interactions with several families of proteins, including p53/p73, NME and GLI. To elucidate the potential interplay among these families we analysed the expression profiles of aforementioned genes and proteins in a panel of melanoma cell lines, metastatic melanoma specimens and healthy corresponding tissue. Using qPCR a higher level of NME1 gene expression and lower levels of Δ40p53ß, ΔNp73, GLI1, GLI2 and PTCH1 were observed in tumour samples compared to healthy tissue. Protein expression of Δ133p53α, Δ160p53α and ΔNp73α isoforms, NME1 and NME2, and N'ΔGLI1, GLI1FL, GLI2ΔN isoforms was elevated in tumour tissue, whereas ∆Np73ß was downregulated. The results in melanoma cell lines, in general, support these findings. In addition, we correlated expression profiles with clinical features and outcome. Higher Δ133p53ß and p53α mRNA and both GLI1 mRNA and GLI3R protein expression had a negative impact on the overall survival. Shorter overall survival was also connected with lower p53ß and NME1 gene expression levels. In conclusion, all examined genes may have implications in melanoma development and functional inactivity of TP53.

Polymorphisms in Survivin (BIRC5 Gene) Are Associated with Age of Onset in Breast Cancer Patients.

Survivin, encoded by BIRC5 gene (baculoviral IAP repeat containing 5), belongs to the family of inhibitors of apoptosis proteins (IAPs). In mammalian cells it participates in the control of mitosis, apoptosis regulation, and cellular stress response. Its expression is increased in almost all types of cancers. The aim of this study was to investigate the role of BIRC5 polymorphisms in breast cancer (BC) and to connect survivin expression with various clinicopathological characteristics of BC patients. Blood and archival tumour tissue samples were collected from 26 BC patients from Croatia. Survivin expression was determined immunohistochemically. BIRC5 promoter, coding region, and 3'UTR were genotyped. DNA from 74 healthy women was used as control. BIRC5 polymorphisms and survivin expression were tested against age of onset, histological grade, tumour type and size, lymph node status, oestrogen, progesterone, Her2, and Ki67 status. Numbers of samples with weak, moderate, and strong survivin expression were 9 (33.3%), 11 (40.7%), and 7 (25.9%), respectively. Most patients had nuclear survivin staining (92.6%). High survivin expression was significantly associated with negative oestrogen receptor status (p=0.007) and positive Ki67 expression (p=0.032). Ki67 expression was also positively correlated with histological grade (p=0.0009). Fourteen polymorphisms were found in BC samples, located mostly in promoter and 3'UTR of BIRC5. There was no significant difference in the distribution of polymorphisms between BC and control samples. Among clinicopathological characteristics of BC patients, alleles of five BIRC5 polymorphisms were associated with younger age of onset: c.-644T>C (55.8 years [y] vs. 48.1 y; p=0.006), c.-241C>T (54.2 y vs. 45.0; p=0.029), c.9809T>C (55.8 y vs. 48.1 y; p=0.006), c.-1547C>T (58.3 y vs. 50.9 y; p=0.011), and c.9386T>C (50.8 y vs. 59.5 y; p=0.004). To assess the significance of BIRC5 polymorphisms and survivin expression as predictive and prognostic biomarkers for BC further research with a larger sample size is needed.

Regulation of Survivin Isoform Expression by GLI Proteins in Ovarian Cancer.

Ovarian cancer (OC) is the most lethal female gynecological malignancy, mostly due to diagnosis in late stages when treatment options are limited. Hedgehog-GLI (HH-GLI) signaling is a major developmental pathway involved in organogenesis and stem cell maintenance, and is activated in OC. One of its targets is survivin (BIRC5), an inhibitor of apoptosis protein (IAP) that plays a role in multiple processes, including proliferation and cell survival. We wanted to investigate the role of different GLI proteins in the regulation of survivin isoform expression (WT, 2α, 2B, 3B, and Δex3) in the SKOV-3 OC cell line. We demonstrated that survivin isoforms are downregulated in GLI1 and GLI2 knock-out cell lines, but not in the GLI3 knock-out. Treatment of GLI1 knock-out cells with GANT-61 shows an additional inhibitory effect on several isoforms. Additionally, we examined the expression of survivin isoforms in OC samples and the potential role of BIRC5 polymorphisms in isoform expression. Clinical samples showed the same pattern of survivin isoform expression as in the cell line, and several BIRC5 polymorphisms showed the correlation with isoform expression. Our results showed that survivin isoforms are regulated both by different GLI proteins and BIRC5 polymorphisms in OC.

Hedgehog-Patched pathway aberrations in a malignant triton tumor case study.

Transition from malignant schwannoma to malignant triton tumor is analyzed in a case report on a patient with recurring cancers and suspected familial predisposition. It is hypothesized that rhabdomyoblastic differentiation, which distinguishes triton from schwannoma, might be attributable to Hedgehog-Patched pathway malfunctioning. Loss of one Patched gene allele was found in the tissue of advanced triton, but the retained allele had no exon or promoter mutations. Protein levels at early cancer stages indicated possible Patched response to the pathway activation in the first occurrence of triton tumor. Later, in the recurring triton, Patched expression was several times lower than in the control tissue, suggesting that haploinsufficiency was aided by silencing of the remaining allele, although its promoter was not hypermethylated. These findings may justify further investigation of the Hedgehog-Patched pathway role in triton malignancies, especially because of the recent research on the therapeutical potential of the pathway.

Analysis of MSH2 Loss of Heterozygosity, Expression, and IVS10+12G>A Polymorphism in Sporadic Colon Cancer.

BACKGROUND: mutS homolog 2 (MSH2) deficiency may be involved in the development of microsatellite instability found in certain sporadic colorectal tumors. In addition to mutations or loss of heterozygosity resulting in complete loss of MSH2 function, polymorphisms affecting MSH2 expression have been also identified. Therefore, the aim of this study was to examine MSH2 status in sporadic colon cancer. MATERIALS AND METHODS: MSH2 status was examined at the DNA, RNA and protein levels through loss of heterozygosity (LOH) analysis, quantitative real-time PCR and immunohistochemistry. MSH2 IVS10+12A>G polymorphism was examined by real-time single nucleotide polymorphism genotyping. RESULTS: MSH2 LOH was more frequent in tumors larger than 5 cm (p=0.032), mRNA expression was also significantly lower and the same expression pattern was present in the corresponding normal mucosa of the same patient (p=0.013 and p=0.008, respectively). No association was found between IVS10+12A>G polymorphism and susceptibility to sporadic colon cancer. CONCLUSION: Altered MSH2 expression detected in sporadic colon tumors pointing to its role in colorectal tumorigenesis without a hereditary component.