Serena Bonin

Inhibition of GSK3B Bypass Drug Resistance of p53-Null Colon Carcinomas by Enabling Necroptosis in Response to Chemotherapy

Purpose: Evasion from chemotherapy-induced apoptosis due to p53 loss strongly contributes to drug resistance. Identification of specific targets for the treatment of drug-resistant p53-null tumors would therefore increase the effectiveness of cancer therapy. Experimental Design: By using a kinase-directed short hairpin RNA library and HCT116p53KO drug-resistant colon carcinoma cells, glycogen synthase kinase 3 beta (GSK3B) was identified as a target whose silencing bypasses drug resistance due to loss of p53. p53-null colon cancer cell lines with different sets of mutations were used to validate the role of GSK3B in sustaining resistance and to characterize cell death mechanisms triggered by chemotherapy when GSK3B is silenced. In vivo xenograft studies were conducted to confirm resensitization of drug-resistant cells to chemotherapy upon GSK3 inhibition. Colon cancer samples from a cohort of 50 chemotherapy-treated stage II patients were analyzed for active GSK3B expression. Results: Downregulation of GSK3B in various drug-resistant p53-null colon cancer cell lines abolished cell viability and colony growth after drug addition without affecting cell proliferation or cell cycle in untreated cells. Cell death of 5-fluorouracil (5FU)–treated p53-null GSK3B-silenced colon carcinoma cells occurred via PARP1-dependent and AIF-mediated but RIP1-independent necroptosis. In vivo studies showed that drug-resistant xenograft tumor mass was significantly reduced only when 5FU was given after GSK3B inhibition. Tissue microarray analysis of colon carcinoma samples from 5FU-treated patients revealed that GSK3B is significantly more activated in drug-resistant versus responsive patients. Conclusions: Targeting GSK3B, in combination with chemotherapy, may represent a novel strategy for the treatment of chemotherapy-resistant tumors. Clin Cancer Res; 19(14); 3820–31. ©2013 AACR.

Management of stage II colon cancer - the use of molecular biomarkers for adjuvant therapy decision

BackgroundThere is uncertainty on the benefit of adjuvant chemotherapy in patients with stage II colorectal cancers. The aim of this study is to investigate the combined role of clinical, pathological and molecular parameters to identify those stage II patients who better benefit from adjuvant therapy.MethodsWe examined 120 stage II colon cancer patients. Of these, 60 patients received adjuvant 5-FU chemotherapy after surgery and the other 60 did not receive therapy. Immunohistochemical (IHC) analyses were performed to evaluate the expressions of Thymidylate synthetase (TYMS), TP53 (p53), β-catenin (CTNNB1) and CD8. For TYMS, its mRNA expression levels were also investigated by real time qRT-PCR. The entire case study was characterized by the presence of a defect in the MMR (mismatch repair) system, the presence of the CpG island methylator phenotype (CIMP or CIMP-High) and for the V600E mutation in the BRAF gene. At the histo-pathological level, the depth of tumour invasion, lymphovascular invasion, invasion of large veins, host lymphocytic response and tumour border configuration were recorded.ResultsThe presence of the V600E mutation in the BRAF gene was a poor prognostic factor for disease free and overall survival (DFS; hazard ratio [HR], 2.57; 95% CI: 1.03 -6.37; p = 0.04 and OS; HR, 3.68; 95% CI: 1.43-9.47; p < 0.01 respectively), independently of 5-FU treatment. Adjuvant therapy significantly improved survival in patients with high TYMS levels (p = 0.04), while patients with low TYMS had a better outcome if treated by surgery alone (DFS; HR, 6.07; 95% CI, 0.82 to 44.89; p = 0.04). In patients with a defect in the MMR system (dMMR), 5-FU therapy was associated to reduced survival (DFS; HR, 37.98; 95% CI, 1.04 to 1381.31; p = 0.04), while it was beneficial for CIMP-High associated tumours (DFS; HR, 0.17; 95% CI, 0.02 to 1.13; p = 0.05).ConclusionsPatients’ characterization according to MMR status, CIMP phenotype and TYMS mRNA expression may provide a more tailored approach for adjuvant therapy in stage II colon cancer.

Detection of Borrelia burgdorferi in skin biopsies from patients with morphea by polymerase chain reaction

Aim We looked for the evidence of Borrelia infection in patients with morphea by serologic means and by polymerase chain reaction (PCR) analysis of skin biopsy samples. Background The possible relationship between Lyme borreliosis and morphea has been suggested by certain clinical, immunological and microbiological findings, but many authors were not be able to demonstrate Borrelia burgdorferi infection in patients with morphea and cast doubts on an etiological role for B. burgdorferi in this skin lesion. Patients and methods Ten patients with morphea, 9 females (range: 8–65 years) and one 44-year-old man were examined. Serological tests for Lyme borreliosis were performed by immunofluorescence assay and flagellin enzyme-linked immunosorbent assay. Skin biopsy specimens were taken from the periphery of morphea lesions for histological examination and PCR. Results Antibodies to B. burgdorferi were detected in 3 patients and B. burgdorferi DNA was demonstrated in 5 patients. Conclusions The amplification of DNA with PCR analysis seems to open new prospects for the detection of Borrelia genome in tissues. In the present study we were able to demonstrate the presence of B. burgdorferi DNA in patients with morphea, even in seronegative patients. These data confirm that PCR is an interesting tool in skin lesion diagnosis and support the hypothesis of an etiological association between B. burgdorferi infection and some cases of morphea.

Detection of Borrelia burgdorferi DNA and complement membrane attack complex deposits in the sural nerve of a patient with chronic polyneuropathy and tertiary lyme disease

We report a patient who developed a chronic sensory motor polyneuropathy and a progressive myelopathy 4 years after a tick bite. An increased serum antibody titer to Borrelia burgdorferi suggested a diagnosis of Lyme neuroborreliosis, although a concomitant cervical spondylosis probably contributed to spinal cord damage. Treatment with ceftriaxone resulted in a marked improvement of neuropathic symptoms, providing indirect evidence of spirochetal infection. Search for B. burgdorferi DNA by polymerase chain reaction amplification on sural nerve confirmed the diagnosis, demonstrating that the spirochete localized in the peripheral nervous system. The presence of complement membrane attack complex deposits and macrophage infiltrates around epineurial vessels and within the endoneurium suggests that the neuropathy in our patient was immune‐mediated.

Higher random oligo concentration improves reverse transcription yield of cDNA from bioptic tissues and quantitative RT-PCR reliability.

Real time quantitative reverse transcription-PCR (qRT-PCR) is the most sensitive technique for detection and quantification of mRNA targets. Reliable quantification of gene expression in formalin-fixed, paraffin-embedded tissues (FFPE), however, has been subjected to serious limitations so far, mainly due to the fragmentation of RNA transcripts. We tried to improve the sensitivity and reliability of mRNA quantification in FFPE by boosting the reverse transcription (RT) step, that is neglected in most of the protocol analysis, but that represents the first confounding event in a quantitative analysis. For this purpose, we compared yield, reproducibility and linearity of RTs performed with random hexamers, random pentadecamers, or a mixture of antisense specific primers in presence of either Moloney murine leukemia virus (MmLV) or the avian myeloblastosis virus (AMV) enzymes. Random primers were tested at two concentrations, 0.14 and 3.35 nmol/reaction. Our qRT-PCR results indicate an improvement of RT yield when using the highest concentration of random oligos with MmLV (from -1.4 to -4.1 C(t)s) in comparison to the lowest concentration. Moreover, more reliable standard curves and therefore, efficiencies were obtained. RT reactions performed with specific primers and AMV were those with the highest yield, but efficiencies were unreliable, due to the RT enzyme-driven PCR inhibition. Random priming at the 3.35 nmol/reaction concentration seems to be the most convenient strategy in assays using RNA obtained from FFPE tissues.

A novel oncogenic BTK isoform is overexpressed in colon cancers and required for RAS-mediated transformation

Bruton’s tyrosine kinase (BTK) is essential for B-cell proliferation/differentiation and it is generally believed that its expression and function are limited to bone marrow-derived cells. Here, we report the identification and characterization of p65BTK, a novel isoform abundantly expressed in colon carcinoma cell lines and tumour tissue samples. p65BTK protein is expressed, through heterogeneous nuclear ribonucleoprotein K (hnRNPK)-dependent and internal ribosome entry site-driven translation, from a transcript containing an alternative first exon in the 5′-untranslated region, and is post-transcriptionally regulated, via hnRNPK, by the mitogen-activated protein kinase (MAPK) pathway. p65BTK is endowed with strong transforming activity that depends on active signal-regulated protein kinases-1/2 (ERK1/2) and its inhibition abolishes RAS transforming activity. Accordingly, p65BTK overexpression in colon cancer tissues correlates with ERK1/2 activation. Moreover, p65BTK inhibition affects growth and survival of colon cancer cells. Our data reveal that BTK, via p65BTK expression, is a novel and powerful oncogene acting downstream of the RAS/MAPK pathway and suggest that its targeting may be a promising therapeutic approach.

Expression of cyclin-dependent kinases and CDC25a phosphatase is related with recurrences and survival in women with peri- and post-menopausal breast cancer

Progression through the mammalian cell cycle is regulated by cyclin—cyclin-dependent kinase (CDKs) complexes that are activated throughout the cell cycle. Alteration in cell cycle control could lead to proliferation and tumourogenesis. This study was designed to analyse, at messenger RNA (mRNA) level, cyclins and CDKs involved in the retinoblastoma pathway, as well as cell division cycle 25a phosphatase (CDC25a), which activates some of the CDKs that were analysed. The aim of the study was to determine the possible prognostic relevance of these molecules in 73 women with peri- and post-menopausal breast cancer. Cyclins A, D1 and E; CDKs 2, 4 and 6 and phosphatase CDC25a expression status were analysed in primary tumours at mRNA level, by reverse transcriptase polymerase chain reaction analysis in paraffin-embedded primary breast cancers. High expression levels of CDK2, CDK4 and CDC25a were related to tumour recurrence. Over-expression of CDK2 and CDC25a was also associated with reduced overall survival; moreover, the CDK2 expression level was able to define a short-living cohort of patients with tumour-positive lymph nodes. CDK2, CDK4 and CDC25a can be used as reliable biomarkers to predict prognosis in women with peri- and post-menopausal breast cancer.

Catalytic subunit of telomerase expression is related to RNA component expression

Telomere length is maintained by the ribonucleoprotein enzyme telomerase. The RNA component of telomerase (hTR) is widespread, and only the expression of the mRNA encoding the catalytic protein subunit (hTRT) is correlated with telomerase activity. We have studied the level of expression of hTR and hTRT in four different models of neoplastic and preneoplastic lesions using the RT‐PCR method on RNA extracted from paraffin‐embedded human tissues after microdissection. The expression at the mRNA level was compared with the enzymatic activity. Our results suggest that there may be a reciprocal control at the transcriptional level of the expression of hTRT and hTR which in turn is associated with tumor progression.

In situ polymerase chain reaction detection of transfusion-transmitted virus in liver biopsy

The potential role of transfusion‐transmitted virus (TTV) infection in determining liver damage is poorly understood and no information exists about TTV replication within hepatocytes. In this study, we assess TTV in situ PCR in liver tissue. Twenty‐one patients with different degrees of liver damage were studied by both serum TTV‐DNA detection and in situ TTV PCR analysis and extractive PCR in liver biopsy paraffin sections (FFPE). Extractive PCR and in situ PCR detected TTV‐DNA both in serum and liver tissue of five patients. The presence of TTV in serum matched with that found in the liver and TTV sequences were never found independently in liver or serum. Four out of five TTV‐DNA‐positive patients have not other known cause of liver damage while in one a coinfection from HCV was observed. Our data indicate that in situ PCR appears to be a reliable tool for the detection of TTV‐DNA in FFPE, and may help detecting unknown origin of liver damage.

Tumour heterogeneity: principles and practical consequences

Two major reasons compel us to study tumour heterogeneity: firstly, it represents the basis of acquired therapy resistance, and secondly, it may be one of the major sources of the low level of reproducibility in clinical cancer research. The present review focuses on the heterogeneity of neoplastic disease, both within the primary tumour and between primary tumour and metastases. We discuss different levels of heterogeneity and the current understanding of the phenomenon, as well as imminent developments relevant for clinical research and diagnostic pathology. It is necessary to develop new tools to study heterogeneity and new biomarkers for heterogeneity. Established and new in situ methods will be very useful. In future studies, not only clonal heterogeneity needs to be addressed but also non-clonal phenotypic heterogeneity which might be important for therapy resistance. We also review heterogeneity established in major tumour types, in order to explore potential similarities that might help to define new strategies for targeted therapy.