Romina Sepe

CBX7 is a tumor suppressor in mice and humans

The CBX7 gene encodes a polycomb group protein that is known to be downregulated in many types of human cancers, although the role of this protein in carcinogenesis remains unclear. To shed light on this issue, we generated mice null for Cbx7. Mouse embryonic fibroblasts derived from these mice had a higher growth rate and reduced susceptibility to senescence compared with their WT counterparts. This was associated with upregulated expression of multiple cell cycle components, including cyclin E, which is known to play a key role in lung carcinogenesis in humans. Adult Cbx7-KO mice developed liver and lung adenomas and carcinomas. In in vivo and in vitro experiments, we demonstrated that CBX7 bound to the CCNE1 promoter in a complex that included HDAC2 and negatively regulated CCNE1 expression. Finally, we found that the lack of CBX7 protein expression in human lung carcinomas correlated with CCNE1 overexpression. These data suggest that CBX7 is a tumor suppressor and that its loss plays a key role in the pathogenesis of cancer.

HMGA1 and HMGA2 protein expression correlates with advanced tumour grade and lymph node metastasis in pancreatic adenocarcinoma

Piscuoglio S, Zlobec I, Pallante P, Sepe R, Esposito F, Zimmermann A, Diamantis I, Terracciano L, Fusco A & Karamitopoulou E 
(2012) Histopathology 60, 397–404 
HMGA1 and HMGA2 protein expression correlates with advanced tumour grade and lymph node metastasis in pancreatic adenocarcinoma

High Mobility Group A Proteins as Tumor Markers

Almost 30 years ago, overexpression of HMGA proteins was associated with malignant phenotype of rat thyroid cells transformed with murine retroviruses. Thereafter, several studies have analyzed HMGA expression in a wide range of human neoplasias. Here, we summarize all these results that, in the large majority of the cases, confirm the association of HMGA overexpression with high malignant phenotype as outlined by chemoresistance, spreading of metastases, and a global poor survival. Even though HMGA proteins’ overexpression indicates a poor prognosis in almost all malignancies, their detection may be particularly useful in determining the prognosis of breast, lung, and colon carcinomas, suggesting for the treatment a more aggressive therapy. In particular, the expression of HMGA2 in lung carcinomas is frequently associated with the presence of metastases. Moreover, recent data revealed that often the cause for the high HMGA proteins levels detected in human malignancies is a deregulated expression of non-coding RNA. Therefore, the HMGA proteins represent tumor markers whose detection can be a valid tool for the diagnosis and prognosis of neoplastic diseases.

CBX7 Modulates the Expression of Genes Critical for Cancer Progression

Background We have previously shown that the expression of CBX7 is drastically decreased in several human carcinomas and that its expression progressively decreases with the appearance of a highly malignant phenotype. The aim of our study has been to investigate the mechanism by which the loss of CBX7 expression may contribute to the emergence of a more malignant phenotype. Methods We analyzed the gene expression profile of a thyroid carcinoma cell line after the restoration of CBX7 and, then, analyzed the transcriptional regulation of identified genes. Finally, we evaluated the expression of CBX7 and regulated genes in a panel of thyroid and lung carcinomas. Results We found that CBX7 negatively or positively regulates the expression of several genes (such as SPP1, SPINK1, STEAP1, and FOS, FOSB, EGR1, respectively) associated to cancer progression, by interacting with their promoter regions and modulating their transcriptional activity. Quantitative RT-PCR analyses in human thyroid and lung carcinoma tissues revealed a negative correlation between CBX7 and its down-regulated genes, while a positive correlation was observed with up-regulated genes. Conclusion In conclusion, the loss of CBX7 expression might play a critical role in advanced stages of carcinogenesis by deregulating the expression of specific effector genes.

UbcH10 overexpression in human lung carcinomas and its correlation with EGFR and p53 mutational status

INTRODUCTION UbcH10 codes for the cancer related E2 Ubiquitin Conjugating Enzyme, an enzymatic molecule with a key role in the ubiquitin-proteasome pathway. Current studies have suggested a critical role of UbcH10 in a variety of malignancies, including human thyroid, breast, ovarian and colorectal carcinomas. The aim of this study has been to extend the analysis of UbcH10 expression to lung cancer. This neoplasia represents one of the leading cause of cancer mortality worldwide, and new tools for an accurate diagnosis/prognosis are needed. METHODS The expression levels of UbcH10 were analysed in human non-small cell lung carcinoma (NSCLC) by quantitative RT-PCR and tissue microarray immunohistochemistry, and these values were correlated with the clinicopathological features of the patients affected by NSCLC. RESULTS Our results demonstrate that UbcH10 is overexpressed in NSCLC compared to the normal lung tissue. Moreover, UbcH10 expression is significantly higher in squamous cell and large cell carcinomas than in adenocarcinomas, and directly and inversely correlated with the mutational status of p53 and EGFR, respectively. The suppression of UbcH10 expression by RNAi resulted in a drastic reduction of proliferation and migration abilities of lung carcinoma cell lines. CONCLUSION These results, taken together, indicate that UbcH10 overexpression has a critical role in lung carcinogenesis, and the evaluation of UbcH10 expression levels may be a new tool for the characterisation of NSCLC.

HMGA1 overexpression is associated with a particular subset of human breast carcinomas

Objectives Breast cancer represents the second leading cause of cancer mortality among American women and accounts for more than 40 000 deaths annually. High-mobility group A1 (HMGA1) expression has been implicated in the pathogenesis and progression of human malignant tumours, including breast carcinomas. The aim of this study was to evaluate HMGA1 detection as an indicator for the diagnosis and prognosis of human breast carcinoma. Methods HMGA1 expression has been analysed by immunohistochemistry in a large series of breast carcinoma resections (1338) combined on a tissue microarray mainly including the ductal carcinoma variant. The results were then correlated with clinicopathological parameters of patients. Results HMGA1 overexpression was found in the large majority of breast carcinoma samples and its overexpression positively correlated with HER-2/neu amplification and progesterone receptor, while a negative correlation was found with oestrogen receptor. Conversely, no HMGA1 expression was found in normal breast tissues. Conclusions The data reported here indicate that HMGA1 is overexpressed in human breast carcinomas and its levels are associated with a particular endocrine status.

KRAS Mutant Allele-Specific Imbalance (MASI) assessment in routine samples of patients with metastatic colorectal cancer

Aims Patients with colorectal cancer harbouring KRAS mutations do not respond to antiepidermal growth factor receptor (anti-EGFR) therapy. Community screening for KRAS mutation selects patients for treatment. When a KRAS mutation is identified by direct sequencing, mutant and wild type alleles are seen on the sequencing electropherograms. KRAS mutant allele-specific imbalance (MASI) occurs when the mutant allele peak is higher than the wild type one. The aims of this study were to verify the rate and tissue distribution of KRAS MASI as well as its clinical relevance. Methods A total of 437 sequencing electropherograms showing KRAS exon 2 mutation was reviewed and in 30 cases next generation sequencing (NGS) was also carried out. Five primary tumours were extensively laser capture microdissected to investigated KRAS MASI tissue spatial distribution. KRAS MASI influence on the overall survival was evaluated in 58 patients. In vitro response to anti-EGFR therapy in relation to different G13D KRAS MASI status was also evaluated. Results On the overall, KRAS MASI occurred in 58/436 cases (12.8%), being more frequently associated with G13D mutation (p=0.05) and having a heterogeneous tissue distribution. KRAS MASI detection by Sanger Sequencing and NGS showed 94% (28/30) concordance. The longer overall survival of KRAS MASI negative patients did not reach statistical significance (p=0.08). In cell line model G13D KRAS MASI conferred resistance to cetuximab treatment. Conclusions KRAS MASI is a significant event in colorectal cancer, specifically associated with G13D mutation, and featuring a heterogeneous spatial distribution, that may have a role to predict the response to EGFR inhibitors. The foreseen implementation of NGS in community KRAS testing may help to define KRAS MASI prognostic and predictive significance.

CBX7 gene expression plays a negative role in adipocyte cell growth and differentiation

ABSTRACT We have recently generated knockout mice for the Cbx7 gene, coding for a polycomb group protein that is downregulated in human malignant neoplasias. These mice develop liver and lung adenomas and carcinomas, which confirms a tumour suppressor role for CBX7. The CBX7 ability to downregulate CCNE1 expression likely accounts for the phenotype of the Cbx7-null mice. Unexpectedly, Cbx7-knockout mice had a higher fat tissue mass than wild-type, suggesting a role of CBX7 in adipogenesis. Consistently, we demonstrate that Cbx7-null mouse embryonic fibroblasts go towards adipocyte differentiation more efficiently than their wild-type counterparts, and this effect is Cbx7 dose-dependent. Similar results were obtained when Cbx7-null embryonic stem cells were induced to differentiate into adipocytes. Conversely, mouse embryonic fibroblasts and human adipose-derived stem cells overexpressing CBX7 show an opposite behaviour. These findings support a negative role of CBX7 in the control of adipocyte cell growth and differentiation.

The cl2/dro1/ccdc80 null mice develop thyroid and ovarian neoplasias.

We have previously reported that the expression of the CL2/CCDC80 gene is downregulated in human papillary thyroid carcinomas, particularly in follicular variants. We have also reported that the restoration of CL2/CCDC80 expression reverted the malignant phenotype of thyroid carcinoma cell lines and that CL2/CCDC80 positively regulated E-cadherin expression, an ability that likely accounts for the role of the CL2/CCDC80 gene in thyroid cancer progression. In order to validate the tumour suppressor role of the CL2/CCDC80 gene in thyroid carcinogenesis we generated cl2/ccdc80 knock-out mice. We found that embryonic fibroblasts from cl2/ccdc80(-/-) mice showed higher proliferation rate and lower susceptibility to apoptosis. Furthermore, cl2/ccdc80(-/-) mice developed thyroid adenomas and ovarian carcinomas. Finally, ret/PTC1 transgenic mice crossed with the cl2/ccdc80 knock-out mice developed more aggressive thyroid carcinomas compared with those observed in the single ret/PTC1 transgenic mice. Together, these results indicate CL2/CCDC80 as a putative tumour suppressor gene in human thyroid carcinogenesis.

UBE2C is overexpressed in ESCC tissues and its abrogation attenuates the malignant phenotype of ESCC cell lines

The esophageal squamous cell carcinoma (ESCC) is widely known as a highly lethal and poor understood cancer, then requiring the search for novel molecular markers to improve its management and patients survival. Recently, ubiquitin-conjugating enzyme E2C (UBE2C) has been figuring as a prominent tumor biomarker candidate, once it has been recognized as a key player in cell cycle progression. In this way, the aim of this study was to evaluate the expression profile of UBE2C gene and protein in ESCC samples, as well as its diagnostic and prognostic marker potential, and its contribution to ESSC genesis and/or progression by performing in vitro functional assays. The analysis of UBE2C gene expression in 52 paired ESCC samples (tumor and respective histologically normal surrounding tissue), by qRT-PCR, revealed that this gene is overexpressed in 73% of ESCC samples. Subsequently, immunohistochemical analysis confirmed that UBE2C protein expression was upregulated in all ESCC cases, but absent in the histologically normal tumor surrounding tissues. Moreover, we showed that UBE2C mRNA expression was able to accurately discriminate ESCC tissue from both healthy esophageal and histologically normal tumor surrounding tissues, pointing out its role as a diagnostic marker for this cancer. Finally, we report that UBE2C affects proliferation rates and cell cycle profile of ESCC cell lines, by directly interfering with cyclin B1 protein levels, suggesting its involvement in crucial steps of ESCC carcinogenesis.