Giosuè Scognamiglio

BAG3 protein is overexpressed in human glioblastoma and is a potential target for therapy.

Glioblastoma multiforme, which represents 80% of malignant gliomas, is characterized by aggressiveness and high recurrence rates. Despite therapeutic advances, patients with glioblastoma multiforme show a poor survival, and identification of novel markers and molecular targets for therapy is needed. A role for BAG3, a member of the BAG family of HSC/HSP70 co-chaperones, in promoting tumor cell growth in vivo has recently been described. We analyzed BAG3 levels by IHC in specimens from patients affected by brain tumors and we found that BAG3, although negative in normal brain tissues, was highly expressed in astrocytic tumors and increasingly expressed in more aggressive types of cancer; it was particularly high in glioblastomas. Down-regulating BAG3 both in vitro and in vivo in a rat glioblastoma model resulted in increased sensitivity to apoptosis, suggesting that BAG3 is a potential target for novel therapies. Finally, we determined that the underlying molecular mechanism requires the formation of a complex of BAG3, HSP70, and BAX that prevents BAX translocation to mitochondria, thus protecting tumor cells from apoptosis. Our data identify BAG3 as a potential marker of glial brain tumor sensitivity to therapy and thus also an attractive candidate for new molecular therapies.

Expression of the Antiapoptotic Protein BAG3 Is a Feature of Pancreatic Adenocarcinoma and Its Overexpression Is Associated With Poorer Survival

Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly cancers, being the fourth leading cause of cancer-related deaths. Long-term survival reaching 15% is achieved in less than 5% of patients who undergo surgery, and median survival is only 6 months in those with inoperable lesions. A deeper understanding of PDAC biologic characteristics as well as novel prognostic markers are therefore required to improve outcomes. Herein we report that BAG3, a protein with recognized anti-apoptotic activity, was expressed in 346 PDACs analyzed, but was not expressed in the surrounding nonneoplastic tissue. In a cohort of 66 patients who underwent radical resection (R0), survival was significantly shorter in patients with high BAG3 expression (median, 12 months) than in those with low BAG3 expression (median, 23 months) (P = 0.001). Furthermore, we report that BAG3 expression in PDAC-derived cell lines protects from apoptosis and confers resistance to gemcitabine, offering a partial explanation for the survival data. Our results indicate that BAG3 has a relevant role in PDAC biology, and suggest that BAG3 expression level might be a potential marker for prediction of patient outcome.

Prognostic value of cancer stem cells, epithelial-mesenchymal transition and circulating tumor cells in lung cancer

The epithelial-mesenchymal transition (EMT) is a program involved in embryonic development that is often activated during cancer invasion and metastasis. CD133 is the main marker identifying cancer stem cells (CSCs) in lung cancer. Circulating tumor cells (CTCs) are demonstrated to be useful as a biomarker for the diagnosis and treatment of cancer. The aim of this study was to correlate EMT, CSCs and CTCs with patient prognosis to verify whether they can contribute to better stratification of lung cancer patients at risk for recurrent and metastatic disease. Pulmonary venous blood was drawn after major pulmonary surgery in 45 patients with resectable non-small cell lung cancer (NSCLC) in order to identify CTCs. For the same patients, we also constructed prognostic lung tissue microarrays (TMA) for CD133 and c-kit and evaluated CSC and EMT markers using flow cytometry. Cytokeratin-positive cells were detectable in 11 (23.9%) cases. c-kit expression was heterogeneous in prognostic TMAs while CD133 expression was low or absent which was also confirmed by flow cytometry and RT-PCR. Flow cytometric analysis showed that the mean percentage of cells with CD133 expression was 1.6%. CD90 and CD326 markers were co-expressed with a mean percentage of 10.41%. When CD133 and CD90/CD326 expression was correlated with follow-up, CD133 showed a higher correlation with deceased patients when compared with CD90/CD326 co-expression (32.5 vs. 9.5%). CD133 expression demonstrated a strong significant association with patients exhibiting progressive disease when compared to CD90/CD326 expression (15 vs. 7.1%). CD133 may be significantly associated with invasion and metastatic spread of NSCLC. The co-expression of CD90, CD326 and CD133 has definite prognostic value in patients with NSCLC.

Role of the cystathionine γ lyase/hydrogen sulfide pathway in human melanoma progression.

In humans, two main metabolic enzymes synthesize hydrogen sulfide (H2S): cystathionine γ lyase (CSE) and cystathionine β synthase (CBS). A third enzyme, 3‐mercaptopyruvate sulfurtransferase (3‐MST), synthesizes H2S in the presence of the substrate 3‐mercaptopyruvate (3‐MP). The immunohistochemistry analysis performed on human melanoma samples demonstrated that CSE expression was highest in primary tumors, decreased in the metastatic lesions and was almost silent in non‐lymph node metastases. The primary role played by CSE was confirmed by the finding that the overexpression of CSE induced spontaneous apoptosis of human melanoma cells. The same effect was achieved using different H2S donors, the most active of which was diallyl trisulfide (DATS). The main pro‐apoptotic mechanisms involved were suppression of nuclear factor‐κB activity and inhibition of AKT and extracellular signal‐regulated kinase pathways. A proof of concept was obtained in vivo using a murine melanoma model. In fact, either l‐cysteine, the CSE substrate, or DATS inhibited tumor growth in mice. In conclusion, we have determined that the l‐cysteine/CSE/H2S pathway is involved in melanoma progression.

Histomorphologic parameters and CXCR4 mRNA and protein expression in sentinel node melanoma metastasis are correlated to clinical outcome.

Introduction: Sentinel lymph node (SLN) biopsy is an important independent prognostic factor for invasive cutaneuos melanoma, although its role is strongly debated. In clinical practice SLN leads to complete lymph node dissection of basin draining melanoma site. However only 7 to 30% of positive sentinel node patients present additional non SLN metastasis. Melanoma cells diffusion through SLN and extranodal spreading depends upon biological features, such as cell chemokine receptors and adhesion molecules. CXCR4 has been proposed in melanoma patients as prognostic marker. Therefore we have analyzed both histopathological parameters and CXCR4 expression in melanoma infiltrate of SLN, in order to evaluate its potential prognostic role. Materials and Methods: 47 positive lymph node metastases were collected and analysed for both histopathological parameters and CXCR4 expression. Results: micrometastases were detected in 23 cases (48.93%); metastases >2mm in 23 cases (48.93%) and isolated metastatic cells in one case (2.01%). High CXCR4 expression was observed in 21 nodal metastases. Node metastases in complete dissection were associated to >10% relative tumor area (RTA) in all lymph nodes (p=0.006). Extranodal invasion (p =0.006) and >2 mm centripetal metastasis thickness (p=0.01), while shorter Disease Free Survival (DFS) was significantly associated to high CXCR4 expression (p=0.02). Conclusion: more than 10% RTA in SLN, extranodal invasion and centripetal metastasis thickness all predict additional lymph node metastases in melanoma site draining basins. Moreover, high CXCR4 expression is correlated to shorter DFS and could be used as a prognostic marker in order to stratify melanoma patients at higher progression risk.

Development and validation of a microRNA-based signature (MiROvaR) to predict early relapse or progression of epithelial ovarian cancer: a cohort study

BACKGROUND Risk of relapse or progression remains high in the treatment of most patients with epithelial ovarian cancer, and development of a molecular predictor could be a valuable tool for stratification of patients by risk. We aimed to develop a microRNA (miRNA)-based molecular classifier that can predict risk of progression or relapse in patients with epithelial ovarian cancer. METHODS We analysed miRNA expression profiles in three cohorts of samples collected at diagnosis. We used 179 samples from a Multicenter Italian Trial in Ovarian cancer trial (cohort OC179) to develop the model and 263 samples from two cancer centres (cohort OC263) and 452 samples from The Cancer Genome Atlas epithelial ovarian cancer series (cohort OC452) to validate the model. The primary clinical endpoint was progression-free survival, and we adapted a semi-supervised prediction method to the miRNA expression profile of OC179 to identify miRNAs that predict risk of progression. We assessed the independent prognostic role of the model using multivariable analysis with a Cox regression model. FINDINGS We identified 35 miRNAs that predicted risk of progression or relapse and used them to create a prognostic model, the 35-miRNA-based predictor of Risk of Ovarian Cancer Relapse or progression (MiROvaR). MiROvaR was able to classify patients in OC179 into a high-risk group (89 patients; median progression-free survival 18 months [95% CI 15-22]) and a low-risk group (90 patients; median progression-free survival 38 months [24-not estimable]; hazard ratio [HR] 1·85 [1·29-2·64], p=0·00082). MiROvaR was a significant predictor of progression in the two validation sets (OC263 HR 3·16, 95% CI 2·33-4·29, p<0·0001; OC452 HR 1·39, 95% CI 1·11-1·74, p=0·0047) and maintained its independent prognostic effect when adjusted for relevant clinical covariates using multivariable analyses (OC179: adjusted HR 1·48, 95% CI 1·03-2·13, p=0·036; OC263: adjusted HR 3·09 [2·24-4·28], p<0·0001; and OC452: HR 1·41 [1·11-1·79], p=0·0047). INTERPRETATION MiROvaR is a potential predictor of epithelial ovarian cancer progression and has prognostic value independent of relevant clinical covariates. MiROvaR warrants further investigation for the development of a clinical-grade prognostic assay. FUNDING AIRC and CARIPLO Foundation.

Deregulation of HOX B13 Expression in Urinary Bladder Cancer Progression

Urinary bladder cancer is a common malignancy in industrialized countries. More than 90% of bladder cancer originates in the transitional cells. Bladder transitional cancer prognosis is, according to the most recent definition related to the level of tumor infiltration, characterized by two main phenotypes, Non Muscle Invasive Bladder Transitional Cancer (NMIBC) and Muscle Invasive Bladder Transitional Cancer (MIBC). The genetic profile and the clinical course of the two subtypes are completely different, however among NMIBC the prognosis is not completely predictable, since 20% of the cases experience a relapse, even in the form of MIBC. It has recently been reported that the chromosomal region 12q13-15, containing crucial cancer genes such as MDM2, CDK4, GLI and an entire cluster of HOX genes, is amplified in bladder cancer. HOX genes codify for transcriptionl factor, involved in embryonal development and cancer progression, with main nuclear expression. Particularly it was also described the strong involvement of HOX B13 in several tumors of urogenital system. In this study we have been investigated, by immunohistochemisty and quantitative Real Time PCR, the HOX B13 expression in bladder cancer evolution and progression, evaluating its ability to discriminate between NMIBC and MBCI phenotypes. Cytoplasmic HOX B13 delocalization significantly relates with muscle invasion (p 0.004). In addition in the series of NMIBC nuclear HOX B13 expression loss is significantly associated to shorter disease free survival (p-value=0.038) defining a potential prognostic role. Overexpression of HOX B13 in more aggressive phenotype is also demonstrate at gene level by quantitative RT-PCR. The de-regulation and delocalization of HOX B13 in urinary bladder cancer supports again the important role of HOX genes in tumor evolution and represents a starting point to establish an integrated analysis, in which HOX genes represent important prognostic and predictive markers for bladder cancer.

Increased HOX C13 expression in metastatic melanoma progression

BackgroundThe process of malignant transformation, progression and metastasis of melanoma is not completely understood. Recently, the microarray technology has been used to survey transcriptional differences that might provide insight into the metastatic process, but the validation of changing gene expression during metastatic transition period is poorly investigated. A large body of literature has been produced on the role of the HOX genes network in tumour evolution, suggesting the involvement of HOX genes in several types of human cancers. Deregulated paralogous group 13 HOX genes expression has been detected in melanoma, cervical cancer and odonthogenic tumors. Among these, Hox C13 is also involved in the expression control of the human keratin genes hHa5 and hHa2, and recently it was identified as a member of human DNA replication complexes.MethodsIn this study, to investigate HOX C13 expression in melanoma progression, we have compared its expression pattern between naevi, primary melanoma and metastasis. In addition HOXC13 profile pattern of expression has been evaluated in melanoma cell lines.ResultsOur results show the strong and progressive HOX C13 overexpression in metastatic melanoma tissues and cytological samples compared to nevi and primary melanoma tissues and cells.ConclusionsThe data presentated in the paper suggest a possible role of HOX C13 in metastatic melanoma switch.

CXCL12-binding receptors expression in non-small cell lung cancer relates to tumoral microvascular density and CXCR4 positive circulating tumoral cells in lung draining venous blood

OBJECTIVES Lung cancer is the main cause of cancer-related death in Western countries. Despite early diagnosis, approximately 40% of patients have undergone surgical resection for localized non-small cell lung cancer relapse within 24 months after surgery. Current prognostic criteria for patients with non-small cell lung cancer are gradually enriched by the discovery of critical biological markers in surgical samples to better stratify patients with high risk for recurrent and metastatic disease after surgical manipulation. In fact, specific biological features are needed to drive metastasis development and, among these chemokine receptors, when activated, seem to play a relevant role, promoting both neovessels formation and tumoral cell migration. METHODS To this purpose, blood samples from the closed stumps of the pulmonary veins were drawn immediately after major pulmonary surgery in 45 patients with resectable non-small cell lung cancer to evaluate the expression of chemokine CXCL12 receptor, CXCR4, in circulating tumor cells. In addition, primary tumor sections have been used to assess microvascular density (MVD) and vessels invasion and build prognostic tissue micro-array to investigate the expression of CXCL12 receptors CXCR4 and CXCR7. RESULTS Cells positive for cytokeratins from tumor draining pulmonary venous blood were detectable in 11 cases (23.9%). In 8 out of 11 cases, CK positive cells coexpressed CXCR4. Moreover, in tumoral tissue high CXCR4 expression was significantly associated to high mMVD (p = 0.046), high CXCR7 expression (p = 0.001), adenocarcinoma histotype (p = 0.023), and to the presence of circulating tumoral cells in pulmonary veins (p = 0.001). Finally, vessel invasions relate to high MVD. CONCLUSION In conclusion, the results of our study underline the significant potential role of CXCL12 receptors in determining both vessel formation and tumoral cell migration to blood stream, favoring metastasis development.

Aberrant Expression of Posterior HOX Genes in Well Differentiated Histotypes of Thyroid Cancers

Molecular etiology of thyroid cancers has been widely studied, and several molecular alterations have been identified mainly associated with follicular and papillary histotypes. However, the molecular bases of the complex pathogenesis of thyroid carcinomas remain poorly understood. HOX genes regulate normal embryonic development, cell differentiation and other critical processes in eukaryotic cell life. Several studies have shown that HOX genes play a role in neoplastic transformation of several human tissues. In particular, the genes belonging to HOX paralogous group 13 seem to hold a relevant role in both tumor development and progression. We have identified a significant prognostic role of HOX D13 in pancreatic cancer and we have recently showed the strong and progressive over-expression of HOX C13 in melanoma metastases and deregulation of HOX B13 expression in bladder cancers. In this study we have investigated, by immunohistochemisty and quantitative Real Time PCR, the HOX paralogous group 13 genes/proteins expression in thyroid cancer evolution and progression, also evaluating its ability to discriminate between main histotypes. Our results showed an aberrant expression, both at gene and protein level, of all members belonging to paralogous group 13 (HOX A13, HOX B13, HOX C13 and HOX D13) in adenoma, papillary and follicular thyroid cancers samples. The data suggest a potential role of HOX paralogous group 13 genes in pathogenesis and differential diagnosis of thyroid cancers.