Long-term in vitro treatment with telomerase inhibitor MST-312 induces resistance by selecting long telomeres cells.

Telomerase is a good target for new anticancer drug development because it is present in over 85% of human tumours. However, despite chronic therapy is a condition for anti-telomerase approach, the effects of long-term treatment with telomerase inhibitors remain not well understood. In this work, it was evaluated the effects of long-term treatment of human MDA-MB-231 breast cancer cells with the telomerase inhibitor MST-312. Cells were treated for 72 hours or 140 days, and it was accessed their viability, proliferation rate, morphology, telomeric DNA content, and resistance mechanism. The drug had a clear short-term effect, including chemosensitizing cells for docetaxel and irinotecan, but the chronic exposition led to selection of long telomeres clones, changing characteristics of original cell line. This effect was confirmed in a clonal culture with homogenous karyotype. MRP-1 expression and alternative lengthening of telomeres (ALT) were discarded as additional mechanisms of resistance. This data suggest that, considering the intra-tumour heterogeneity (ITH), what is already a big challenge for treatment of cancer, chronic exposition to telomerase inhibitors can promote tumour adaptations with potential clinical repercussion, drawing attention to ongoing clinical trials and pointing important considerations most times neglected on studies about use of these inhibitors on cancer therapy. SIGNIFICANCE OF THE STUDY: Antitumour action of telomerase inhibitors is well known, but it depends on a long-term exposition because cells will undergo telomere erosion only after many duplication cycles. Recently, the frustrating results of clinical trials with these inhibitors aroused the interest of the scientific community to understand the mechanisms of resistance to anti-telomerase therapy. In this study, we conducted an 18-week experiment to show that telomerase inhibition can lead to cell adaptations and selection of long-telomeres clones, leading to acquisition of resistance. However, we also showed that this inhibitor can sensitize cells to the chemotherapeutic drugs docetaxel and irinotecan.

The classical photoactivated drug 8-methoxypsoralen and related compounds are effective without UV light irradiation against glioma cells.

Currently, there is no effective therapy for high grade gliomas. 8-Methoxypsoralen (8-MOP) is a compound used in the treatment of skin diseases combined with UV light irradiation. In this work, rat glioma C6 cells, normal astrocytes and human glioblastoma GL-15 cells comprised an in vitro model to evaluate the antitumor activity of 8-MOP. We found that 8-MOP promoted a time- and concentration-dependent reduction of cell viability in tumor, but not in normal cells. This effect was more evident in log-phase growing culture, indicating antiproliferative activity, which was confirmed by colony formation assay. Long-term effect of 8-MOP at low concentration was also attested. The concentrations used in the tests (0.02-0.4 mM) were lower than plasmatic concentration found in patients. Despite the treatment leads to considerable morphological changes and apoptosis when used at high concentrations, 8-MOP did not promote cell cycle arrest, change in migration pattern neither necrosis. In addition, we evaluated the effect of 8-MOP in MDA-MB-231, CT-26 and SCC-3 cell lines, derived from other kind of primary tumors, and found that CT-26 cells did not respond to 8-MOP treatment, indicating that this compound does not act through a generic mechanism. Coumarin derivatives structurally related to 8-MOP were screened for its antitumor potential and presented different patterns of biological activity, and then it was possible to suggest the relevance of 8-MOP molecular structure for antiproliferative action. Therefore, 8-MOP, a drug with an outstanding record of safety, and related coumarins are good prototypes for development of a new class of anti-glioma drugs.

ERBB2 gene as a potential therapeutic target in small bowel adenocarcinoma.

AIM OF THE STUDY: Small bowel adenocarcinoma (SBA) is a rare and aggressive tumour with poor outcomes. Because of its low incidence, the number prospective studies remains insufficient leading to poor knowledge and absence of standard of care. Aiming to better understand small bowel carcinogenesis we investigated the frequency of somatic mutations in a large data set of patients in more than 740 mutational hotspots among 46 genes. METHODS: In total, 83 SBA cases were selected from two European databases. The sequencing was performed using the Ion 316 Chip. Additionally we looked into ERBB2 expression and microsatellite instability (MSI) status. RESULTS: The tumours most frequently were duodenal (47%) and stage ⩾3 (63%). Eight genes were mutated with a frequency >5%: KRAS, TP53, APC, SMAD4, PIK3CA, ERBB2, BRAF and FBXW7. ERBB2 alterations are present in 10 patients (12%) through mutations (7 cases) or amplifications (3 cases). ERBB2 mutations were significantly associated with duodenal tumour location (P=0.04). In this group, there was a positive association with dMMR status (P=0.006) and APC mutation (P=0.02) but negative association with p53 mutations (P=0.038). CONCLUSIONS: This study describes the first large screening of somatic mutations in SBA using next generation sequencing. The ERBB2 mutation was revealed to be one of the most frequent alterations in SBA with a distribution dependent on tumour location. In most cases ERBB2 mutation was identical (p.L755S). In clinical practice, this may suggest that more than 10% of the patients with SBA could be treated using an anti-ERBB2-targeted agent.

Altered expression of MLL methyltransferase family genes in breast cancer.

The histone lysine methyltransferases contain a SET domain, which catalyzes the addition of methyl groups to specific lysine residues. The MLL family of genes encodes histone-modifying enzymes with histone 3-lysine 4 methyltransferase activity that can regulate gene transcription. The MLL family exists in multi-protein complexes and has been implicated in a variety of processes including normal development and cell growth. Although some of the MLL family members have already been described to be involved in cancer, a clear relationship of these genes with breast cancer is not determined to date. In the present study, we used quantitative PCR to investigate the expression profile of all five MLL genes [MLL (ALL-1), MLL2, MLL3, MLL4 and MLL5] in 7 breast cancer cell lines, 8 breast tumors and adjacent non-tumor tissues and in 12 normal tissues. We observed a diminished expression of all five genes in the breast cancer cell lines when compared to normal breast tissue. We found a significantly decreased expression of MLL2 in the tumor samples compared to the non-tumor controls. In tumor samples, MLL5 also showed a clear suppression tendency. Among the normal tissues analyzed, all genes showed a markedly higher expression in skeletal muscle and brain. Although further studies are required to determine the exact role of these methyltransferases in cancer development, our results indicate that the suppression of MLL genes, especially MLL2 and 5, take part in modulating breast carcinogenesis. Our assessment of the MLL family gene expression patterns in a diverse set of breast cancer cell lines and in a multitude of tissue types and breast tumors should lead to increasingly detailed information on the involvement of these genes in cancer progression.

SET domain-containing Protein 4 (SETD4) is a Newly Identified Cytosolic and Nuclear Lysine Methyltransferase involved in Breast Cancer Cell Proliferation.

Cancer is comprised of a multitude of epigenetic abnormalities, including the global loss and regional gain of DNA methylation as well as alterations in histone methylation. Here, we characterize a new methyltransferase, SET domain-containing protein 4 (SETD4), which is involved in breast carcinogenesis. Quantitative real-time PCR (qPCR) showed elevated expression levels of SETD4 in several breast cancer cell lines. SETD4 overexpression was confirmed by western blot analysis suggesting a correlation between high expression of SETD4 and a lack of the estrogen receptor (ER) in breast cancer. In addition, cell fractionation studies and confocal immunofluorescence revealed the nuclear and non-nuclear localization of this new protein. SETD4 knockdown in breast cancer cell lines significantly suppressed their proliferation and delayed the G1/S cell cycle transition without affecting apoptosis. Furthermore, western blot analysis showed that knockdown of SETD4 decreased cyclin D1 expression, revealing the involvement of SETD4 in cell cycle regulation. These data imply that SETD4 plays a crucial role in breast carcinogenesis and could be a novel molecular target for the development of new strategies for the diagnosis and treatment of breast cancer.

The lysine 831 of vascular endothelial growth factor receptor 1 is a novel target of methylation by SMYD3.

We previously identified SMYD3 as a histone methyltransferase and showed that its expression was elevated in colorectal, hepatocellular, and breast carcinomas. In the investigation of methyltransferase activity of SMYD3, we have found that vascular endothelial growth factor receptor 1 (VEGFR1) was also methylated by SMYD3. We further identified the methylated residue at VEGFR1 lysine 831, which is located in the kinase domain and is conserved among VEGFR1 orthologues. We also found that the lysine is followed by serine, which is conserved among some of the methylation targets of histone methyltransferases. Furthermore, methylation of VEGFR1 enhanced its kinase activity in cells. These data should be helpful for the profound understanding of the biological role of SMYD3 and regulatory mechanisms of VEGFR1. Additionally our finding may facilitate the development of strategies that may inhibit the progression of cancer cells.

Enhanced SMYD3 expression is essential for the growth of breast cancer cells.

We previously reported that upregulation of SMYD3, a histone H3 lysine-4-specific methyltransferase, plays a key role in the proliferation of colorectal carcinoma (CRC) and hepatocellular carcinoma (HCC). In the present study, we reveal that SMYD3 expression is also elevated in the great majority of breast cancer tissues. Similarly to CRC and HCC, silencing of SMYD3 by small interfering RNA to this gene resulted in the inhibited growth of breast cancer cells, suggesting that increased SMYD3 expression is also essential for the proliferation of breast cancer cells. Moreover, we show here that SMYD3 could promote breast carcinogenesis by directly regulating expression of the proto-oncogene WNT10B. These data imply that augmented SMYD3 expression plays a crucial role in breast carcinogenesis, and that inhibition of SMYD3 should be a novel therapeutic strategy for treatment of breast cancer.

A variable number of tandem repeats polymorphism in an E2F-1 binding element in the 5' flanking region of SMYD3 is a risk factor for human cancers.

Histone modification is a crucial step in transcriptional regulation, and deregulation of the modification process is important in human carcinogenesis. We previously reported that upregulation of SMYD3, a histone methyltransferase, promoted cell growth in human colorectal and hepatocellular carcinomas. Here we report significant associations between homozygosity with respect to an allele with three tandem repeats of a CCGCC unit in the regulatory region of SMYD3 and increased risk of colorectal cancer (P = 9.1 x 10(-6), odds ratio = 2.58), hepatocellular carcinoma (P = 2.3 x 10(-8), odds ratio = 3.50) and breast cancer (P = 7.0 x 10(-10), odds ratio = 4.48). This tandem-repeat sequence is a binding site for the transcriptional factor E2F-1. In a reporter assay, plasmids containing three repeats of the binding motif (corresponding to the high-risk allele) had higher activity than plasmids containing two repeats (the low-risk allele). These data suggest that the common variable number of tandem repeats polymorphism in SMYD3 is a susceptibility factor for some types of human cancer.

WDRPUH, a novel WD-repeat-containing protein, is highly expressed in human hepatocellular carcinoma and involved in cell proliferation.

In an attempt to disclose mechanisms of hepatocarcinogenesis and discover novel target molecules for the diagnosis and treatment of hepatocellular carcinomas (HCCs), we previously analyzed expression profiles of HCC tissues by means of human cDNA microarray. Among the genes upregulated in tumor tissues compared with their nontumor counterparts, we focused on a novel gene, termed WDRPUH, and characterized its biologic function. WDRPUH encodes a predicted 620-amino acid protein containing 11 highly conserved WD40-repeat domains. Multiple-tissue Northern blot analysis revealed its specific expression in the testis among 16 normal tissues examined. Transfection of plasmids designed to express WDRPUH-specific siRNA significantly reduced its expression in HCC cells and resulted in growth suppression of transfected cells. Interestingly, we found that WDRPUH associated with HSP70, proteins of the chaperonin-containing TCP-1 (CCT1) complex, as well as BRCA2. These findings have disclosed a novel insight into hepatocarcinogenesis and suggested that WDRPUH may be a molecular target for the development of new strategies to treat HCCs.

SMYD3 encodes a histone methyltransferase involved in the proliferation of cancer cells.

Colorectal and hepatocellular carcinomas are some of the leading causes of cancer deaths worldwide, but the mechanisms that underly these malignancies are not fully understood. Here we report the identification of SMYD3, a gene that is over-expressed in the majority of colorectal carcinomas and hepatocellular carcinomas. Introduction of SMYD3 into NIH3T3 cells enhanced cell growth, whereas genetic knockdown with small-interfering RNAs (siRNAs) in cancer cells resulted in significant growth suppression. SMYD3 formed a complex with RNA polymerase II through an interaction with the RNA helicase HELZ and transactivated a set of genes that included oncogenes, homeobox genes and genes associated with cell-cycle regulation. SMYD3 bound to a motif, 5'-CCCTCC-3', present in the promoter region of downstream genes such as Nkx2.8. The SET domain of SMYD3 showed histone H3-lysine 4 (H3-K4)-specific methyltransferase activity, which was enhanced in the presence of the heat-shock protein HSP90A. Our findings suggest that SMYD3 has histone methyltransferase activity and plays an important role in transcriptional regulation as a member of an RNA polymerase complex. Furthermore, activation of SMYD3 may be a key factor in human carcinogenesis.