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.

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.

Tumor markers in breast cancer--evaluation of their clinical usefulness.

Breast cancer is the most common neoplasm affecting women in the Western world. Many studies are still conducted with the purpose of finding markers that could be used for early diagnosis and/or serve as possible reliable prognostic or predictive parameters, but with conflicting results. At present, no markers are available for an early diagnosis of breast cancer For surveillance of patients with diagnosed breast cancer the most widely used serum markers are CA 15-3 and CEA which, in combination with other clinical parameters, could have clinical significance. The most useful and clinically important tissue-based markers in breast cancer are estrogen and progesterone receptors, used as a basis for hormonal therapy, and HER-2 receptors, essential in selecting patients for the treatment with Herceptin. New or potentially new markers for breast cancer include BRCA1 and BRCA2 genes for selecting patients at high risk of developing hereditary breast cancer, as well as urokinase plasminogen activator and inhibitor for assessing prognosis in lymph node-negative patients. Results of tumor and patient genetic analyses including their clinical evaluation will enable application of more individualized and personalized approach in diagnosis and therapy of breast cancer patients.

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.

[Molecular basis of breast cancer related to BRCA 1 and BRCA2 genes: characteristics and targeting therapy]. / Molekularna osnova raka dojke vezana uz gene BRCA1 i BRCA2: karakteristike i ciljana terapija.

Breast cancer is one of the most frequent tumors in women, and BRCA1 and BRCA2 genes play a major role in the hereditary susceptibility for this disease. Until the age of 70 women carrying a mutation in BRCA1 or BRCA2 gene have a 45-85% probability of developing breast cancer, and 11-62% probability of developing ovarian cancer. Mutation carrier's tumors contain nonfunctional BRCA1 or BRCA2 genes, which in healthy cells are involved in DNA repair. These tumors show an increased sensitivity to DNA damaging chemical agents and to PARP (poly(adenosine diphosphate-ribose) polymerase1) inhibitors. New targeted therapies already in use are directed toward tumors of mutation carriers. Successful treatment is most likely to be achieved through cooperation of a pathologist, oncologist and a genetic laboratory performing BRCA genes mutation screening.

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.

The Patched gene is epigenetically regulated in ovarian dermoids and fibromas, but not in basocellular carcinomas.

The Hedgehog/Patched signaling pathway plays a prominent role during mammalian development but it is also involved in oncogenic transformation. We investigated the methylation status of the Patched promotor in a set of basocellular carcinomas of the skin and ovarian tumors as an alternative to mutational causes of the pathway deregulation. Our aim was to define a possible role of genetic and/or epigenetic mechanisms of Hedgehog/Patched signal transduction in the development of these tumors. Bisulfite-converted DNA from tumors and from matched healthy tissue was amplified by a specific PCR and the CpG-rich regions of the Patched promoter were sequenced. Two promoter regions showed statistically significant hypermethylation compared to healthy controls in ovarian tumors; more significantly in the region in the vicinity of Gli1-binding sites and less significantly in the region containing the ATG codon. But, in basocellular carcinomas of the skin we observed no difference in methylation, suggesting different mechanisms of neoplasia in these tumors.

The first case report of a large deletion of the BRCA1 gene in Croatia: A case report.

RATIONALE: Breast cancer is one of the most common cancers in women, and it is the leading cause of cancer related deaths in Croatia. BRCA1 and BRCA2 gene mutations are the most common cause of hereditary breast cancer. PATIENT CONCERNS: In this report we describe a Croatian patient with no apparent family history of cancer, who developed breast cancer first at 29, and again at 33. DIAGNOSIS: Due to the early development of first breast cancer and triple negative status of the second, the attending physician suspected a hereditary aspect. INTERVENTIONS: Patient was sent to BRCA1 genetic testing. Subsequently, her mother and sister were sent to check for the mutation found in the patient. OUTCOMES: BRCA1 exons 4-6 deletion was determined and sequencing confirmed the deletion as NG_005905.2:g.107648_117905del10257. Mother and sister were not affected, but since there were no available family members on the fathers' side, it was not possible to determine if this was a case of de novo mutation. Until now, only in three reports with the similar mutation the exact mutation borders were determined. The mutation in this case was not the same as previously reported and was more than twice in size. LESSONS: All large deletions should be described at the nucleotide level, so that in cases with missing family data it would be possible to deduce if the mutation is already known. If the mutation is already known, it is probably not a de novo event, since it is unlikely that the breakpoints would be exactly the same more than once.

Hedgehog Signaling Pathway as Genetic and Epigenetic Target in Ovarian Tumors.

BACKGROUND: Hedgehog signaling pathway is a developmental pathway mostly inactive in adult tissues, with the exception of stem cells. It is often found upregulated in various tumors, and associated with cancer stem cell maintenance. METHODS: This review focuses on different aspects of Hedgehog activation in tumors, with special emphasis on ovarian tumors and their treatment. RESULTS: Mutations in pathway components lead to a series of developmental malformations and syndromes. Aberrant activation of the pathway can be caused by mutations, noncanonical transcriptional regulation, or epigenetic changes. CONCLUSION: This pathway is an interesting target in cancer therapy, especially when combined with therapies targeting other signaling pathways. Combination therapy can be used to bypass resistance or to target cancer stem cells in a more efficient way.

Non-canonical Hedgehog signaling activation in ovarian borderline tumors and ovarian carcinomas.

Hedgehog signaling pathway has been implicated in the pathology of ovarian cancer, and Survivin (BIRC5) has been suggested as a novel target of this pathway. Herein we investigated the role of Hedgehog signaling pathway and Survivin in ovarian carcinoma and borderline tumor samples. We aimed to determine possible ways of pathway modulation on primary ovarian cancer cells and an established cell line. RNA was extracted from fresh tumors and control tissues and gene expression was examined using qRT-PCR. Pathway activity in cell lines was examined after treatment with cyclopamine, SHH protein, GANT-61 or lithium chloride using qRT-PCR, western blot and confocal microscopy. The difference between control tissue, borderline tumors and carcinomas can be seen in GLI1 and SUFU gene expression, which is significantly higher in borderline tumors compared to carcinomas. SUFU also shows lower expression levels in higher FIGO stages relative to lower stages. BIRC5 is expressed in all tumors and in healthy ovarian tissues compared to our control tissue, healthy fallopian tube samples. Primary cells developed from ovarian carcinoma tissue respond to cyclopamine treatment with a short-term decrease in cell proliferation, downregulation of Hedgehog pathway genes, including BIRC5, and changes in protein dynamics. Stimulation with SHH protein results in increased cell migration, while GLI1 transfection or PTCH1 silencing demonstrate pathway upregulation. The pathway activity can be modulated by LiCl at the GSK3ß-SUFU-GLI level, suggesting at least partial non-canonical activation. Downregulation of the pathway with GANT-61 has proved to be more effective than cyclopamine. GLI inhibitors may be a superior treatment option in ovarian cancer compared to SMO inhibitors.