Paola Nisticò

Epithelial-Mesenchymal Transition: General Principles and Pathological Relevance with Special Emphasis on the Role of Matrix Metalloproteinases

Epithelial-mesenchymal transition (EMT) is a physiological process in which epithelial cells acquire the motile and invasive characteristics of mesenchymal cells. Although EMT in embryonic development is a coordinated, organized process involving interaction between many different cells and tissue types, aspects of the EMT program can be inappropriately activated in response to microenvironmental alterations and aberrant stimuli, and this can contribute to disease conditions including tissue fibrosis and cancer progression. Here we will outline how EMT functions in normal development, how it could be activated in pathologic conditions-especially by matrix metalloproteinases-and how it may be targeted for therapeutic benefit.

Human mena protein, a serex‐defined antigen overexpressed in breast cancer eliciting both humoral and CD8+ T‐cell immune response

Screening of a cDNA expression library from a primary breast tumor with the autologous patient serum led to the isolation of 6 cDNA clones corresponding to 3 different genes, including a novel gene that maps to chromosome 1 and encodes the human homologue of mouse Mena (hMena, cDNA clone RMNY‐BR‐55), a protein of the Ena/VASP family involved in the regulation of cell motility and adhesion. A cancer‐restricted antibody response against hMena was demonstrated, since 18/93 cancer patient sera, the majority (10/52) from breast cancer, showed anti‐hMena‐specific IgG, while no antibodies were present in healthy donors. When hMena protein expression was analyzed by Western blot and immunohistochemistry, the antigen was overexpressed in the majority of breast cancer cell lines and in 75% of primary breast tumor lesions evaluated. Furthermore, when HLA‐A2‐restricted peptides from the hMena sequence were used to stimulate CD8+ T cells, an hMena‐specific response was found in 9 out of 12 HLA‐A2+ breast cancer patients. In 4 patients, this cell‐mediated immune response was concomitant with antibody response to hMena. Furthermore, an hMena‐specific T‐cell line was established from an HLA‐A2+ breast cancer patient whose primary tumor lesion overexpressed the hMena protein. The present findings highlight the emerging role that overexpression of cytoskeleton regulatory components may have in the induction of a specific antitumor immune response.

Chemotherapy enhances vaccine-induced antitumor immunity in melanoma patients.

Combination of chemotherapy with cancer vaccines is currently regarded as a potentially valuable therapeutic approach for the treatment of some metastatic tumors, but optimal modalities remain unknown. We designed a phase I/II pilot study for evaluating the effects of dacarbazine (DTIC) on the immune response in HLA‐A2+ disease‐free melanoma patients who received anticancer vaccination 1 day following chemotherapy (800 mg/mq i.v.). The vaccine, consisting of a combination of HLA‐A2 restricted melanoma antigen A (Melan‐A/MART‐1) and gp100 analog peptides (250 μg each, i.d.), was administered in combination or not with DTIC to 2 patient groups. The combined treatment is nontoxic. The comparative immune monitoring demonstrates that patients receiving DTIC 1 day before the vaccination have a significantly improved long‐lasting memory CD8+ T cell response. Of relevance, these CD8+ T cells recognize and lyse HLA‐A2+/Melan‐A+ tumor cell lines. Global transcriptional analysis of peripheral blood mononuclear cells (PBMC) revealed a DTIC‐induced activation of genes involved in cytokine production, leukocyte activation, immune response and cell motility that can favorably condition tumor antigen‐specific CD8+ T cell responses. This study represents a proof in humans of a chemotherapy‐induced enhancement of CD8+ memory T cell response to cancer vaccines, which opens new opportunities to design novel effective combined therapies improving cancer vaccination effectiveness.

Circulating Autoantibodies to Phosphorylated α-Enolase are a Hallmark of Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis and no diagnostic markers have, as of yet, been defined. In PDAC patients, α-enolase (ENOA) is up-regulated and elicits the production of autoantibodies. Here, we analyzed the autoantibody response to post-translational modifications of ENOA in PDAC patients. ENOA isolated from PDAC tissues and cell lines was characterized by two-dimensional electrophoresis (2-DE) Western blot (WB), revealing the expression of six different isoforms (named ENOA1,2,3,4,5,6) whereas only 4 isoforms (ENOA3,4,5,6) were detectable in normal tissues. As assessed by 2-DE WB, 62% of PDAC patients produced autoantibodies to the two more acidic isoforms (ENOA1,2) as opposed to only 4% of controls. Mass spectrometry showed that ENOA1,2 isoforms were phosphorylated on serine 419. ROC analysis demonstrated that autoantibodies to ENOA1,2 usefully complement the diagnostic performance of serum CA19.9 levels, achieving approximately 95% diagnostic accuracy in both advanced and resectable PDAC. Moreover, the presence of autoantibodies against ENOA1,2 correlated with a significantly better clinical outcome in advanced patients treated with standard chemotherapy. In conclusion, our results demonstrate that ENOA phosphorylation is associated with PDAC and induces specific autoantibody production in PDAC patients that may have diagnostic value.

Molecular Cloning of hMena (ENAH) and Its Splice Variant hMena+11a: Epidermal Growth Factor Increases Their Expression and Stimulates hMena+11a Phosphorylation in Breast Cancer Cell Lines

hMena (ENAH), an actin regulatory protein involved in the control of cell motility and adhesion, is modulated during human breast carcinogenesis. In fact, whereas undetectable in normal mammary epithelium, hMena becomes overexpressed in high-risk benign lesions and primary and metastatic tumors. In vivo, hMena overexpression correlates with the HER-2(+)/ER(-)/Ki67(+) unfavorable prognostic phenotype. In vitro, neuregulin-1 up-regulates whereas Herceptin treatment down-modulates hMena expression, suggesting that it may couple tyrosine kinase receptor signaling to the actin cytoskeleton. Herein, we report the cloning of hMena and of a splice variant, hMena(+11a), which contains an additional exon corresponding to 21 amino acids located in the EVH2 domain, from a breast carcinoma cell line of epithelial phenotype. Whereas hMena overexpression consistently characterizes the transformed phenotype of tumor cells of different lineages, hMena(+11a) isoform is concomitantly present only in epithelial tumor cell lines. In breast cancer cell lines, epidermal growth factor (EGF) treatment promotes concomitant up-regulation of hMena and hMena(+11a), resulting in an increase of the fraction of phosphorylated hMena(+11a) isoform only. hMena(+11a) overexpression and phosphorylation leads to increased p42/44 mitogen-activated protein kinase (MAPK) activation and cell proliferation as evidenced in hMena(+11a)-transfected breast cancer cell lines. On the contrary, hMena knockdown induces reduction of p42/44 MAPK phosphorylation and of the proliferative response to EGF. The present data provide new insight into the relevance of actin cytoskeleton regulatory proteins and, in particular, of hMena isoforms in coupling multiple signaling pathways involved in breast cancer.

An integrated humoral and cellular response is elicited in pancreatic cancer by α-enolase, a novel pancreatic ductal adenocarcinoma-associated antigen†

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease with a very poor 5‐year survival rate. α‐Enolase is a glycolytic enzyme that also acts as a surface plasminogen receptor. We find that it is overexpressed in PDAC and present on the cell surface of PDAC cell lines. The clinical correlation of its expression with tumor status has been reported for lung and hepatocellular carcinoma. We have previously demonstrated that sera from PDAC patients contain IgG autoantibodies to α‐enolase. The present work was intended to assess the ability of α‐enolase to induce antigen‐specific T cell responses. We show that α‐enolase‐pulsed dendritic cells (DC) specifically stimulate healthy autologous T cells to proliferate, secrete IFN‐γ and lyse PDAC cells but not normal cells. In vivo, α‐enolase‐specific T cells inhibited the growth of PDAC cells in immunodeficient mice. In 8 out of 12 PDAC patients with circulating IgG to α‐enolase, the existence of α‐enolase‐specific T cells was also demonstrated. Taken as a whole, these results indicate that α‐enolase elicits a PDAC‐specific, integrated humoral and cellular response. It is thus a promising and clinically relevant molecular target candidate for immunotherapeutic approaches as new adjuvants to conventional treatments in pancreatic cancer.

Splicing program of human MENA produces a previously undescribed isoform associated with invasive, mesenchymal-like breast tumors

Human mena (hMENA), a member of the actin cytoskeleton regulators Ena/VASP, is overexpressed in high-risk preneoplastic lesions and in primary breast tumors and has been identified as playing a role in invasiveness and poor prognosis in breast cancers that express HER2. Here we identify a unique isoform, hMENAΔv6, derived from the hMENA alternative splicing program. In an isogenic model of human breast cancer progression, we show that hMENA11a is expressed in premalignant cells, whereas hMENAΔv6 expression is restricted to invasive cancer cells. “Reversion” of the malignant phenotype leads to concurrent down-regulation of all hMENA isoforms. In breast cancer cell lines, isoform-specific hMENA overexpression or knockdown revealed that in the absence of hMENA11a, overexpression of hMENAΔv6 increased cell invasion, whereas overexpression of hMENA11a reduced the migratory and invasive ability of these cells. hMENA11a splicing was shown to be dependent on the epithelial regulator of splicing 1 (ESRP1), and forced expression of ESRP1 in invasive mesenchymal breast cancer cells caused a phenotypic switch reminiscent of a mesenchymal-to-epithelial transition (MET) characterized by changes in the cytoskeletal architecture, reexpression of hMENA11a, and a reduction in cell invasion. hMENA-positive primary breast tumors, which are hMENA11a-negative, are more frequently E-cadherin low in comparison with tumors expressing hMENA11a. These data suggest that polarized and growth-arrested cellular architecture correlates with absence of alternative hMENA isoform expression, and that the hMENA splicing program is relevant to malignant progression in invasive disease.

Human Mena+11a isoform serves as a marker of epithelial phenotype and sensitivity to epidermal growth factor receptor inhibition in human pancreatic cancer cell lines.

Purpose: hMena, member of the enabled/vasodilator-stimulated phosphoprotein family, is a cytoskeletal protein that is involved in the regulation of cell motility and adhesion. The aim of this study was to determine whether or not the expression of hMena isoforms correlated with sensitivity to EGFR tyrosine kinase inhibitors and could serve as markers with potential clinical use. Experimental Design: Human pancreatic ductal adenocarcinoma cell lines were characterized for in vitro sensitivity to erlotinib, expression of HER family receptors, markers of epithelial to mesenchymal transition, and expression of hMena and its isoform hMena+11a. The effects of epidermal growth factor (EGF) and erlotinib on hMena expression as well as the effect of hMena knockdown on cell proliferation were also evaluated. Results: hMena was detected in all of the pancreatic tumor cell lines tested as well as in the majority of the human tumor samples [primary (92%) and metastatic (86%)]. Intriguingly, in vitro hMena+11a isoform was specifically associated with an epithelial phenotype, EGFR dependency, and sensitivity to erlotinib. In epithelial BxPC3 cells, epidermal growth factor up-regulated hMena/hMena+11a and erlotinib down-regulated expression. hMena knockdown reduced cell proliferation and mitogen-activated protein kinase and AKT activation in BxPC3 cells, and promoted the growth inhibitory effects of erlotinib. Conclusions: Collectively, our data indicate that the hMena+11a isoform is associated with an epithelial phenotype and identifies EGFR-dependent cell lines that are sensitive to the EGFR inhibitor erlotinib. The availability of anti-hMena+11a–specific probes may offer a new tool in pancreatic cancer management if these results can be verified prospectively in cancer patients.

The Cytoskeleton Regulatory Protein hMena (ENAH) Is Overexpressed in Human Benign Breast Lesions with High Risk of Transformation and Human Epidermal Growth Factor Receptor-2–Positive/Hormonal Receptor–Negative Tumors

Purpose: hMena (ENAH), a cytoskeleton regulatory protein involved in the regulation of cell motility and adhesion, is overexpressed in breast cancer. The aim of this study was to define at what stage of breast carcinogenesis hMena is overexpressed and to correlate hMena overexpression with established prognostic factors in breast cancer, focusing on human epidermal growth factor receptor-2 (HER-2). Experimental Design: hMena expression was assessed immunohistochemically in a prospective cohort of cases (n = 360) encompassing a highly representative spectrum of benign breast diseases associated with different risk of transformation, in situ, invasive, and metastatic tumors. Correlations with conventional pathologic and prognostic variables, such as proliferation index, hormonal receptor status, and HER-2 overexpression, were also evaluated. In vitro experiments were done to study the effect of neuregulin-1 and Herceptin treatments on hMena expression. Results: hMena protein is undetectable in normal breast and is weakly expressed in a small percentage of low-risk benign diseases (9%), but displays a progressive and significant increase of positivity in benign lesions at higher risk of transformation (slightly increased risk 43%; moderate increased risk 67%), in in situ (72%), invasive (93%), and metastatic breast cancer (91%). A significant direct correlation with tumor size (P = 0.04), proliferation index (P < 0.0001), and HER-2 overexpression (P < 0.0001) and an inverse relationship with estrogen (P = 0.036) and progesterone receptors (P = 0.001) are found in invasive carcinomas. In vitro experiments show that neuregulin-1 up-regulates, whereas Herceptin down-regulates, hMena expression. Conclusions: Our data provide new insights into the relevance of actin-binding proteins in human breast carcinogenesis and indicate hMena overexpression as a surrogate indicator in breast disease management.

Dacarbazine Treatment before Peptide Vaccination Enlarges T-Cell Repertoire Diversity of Melan-A―Specific, Tumor-Reactive CTL in Melanoma Patients

Combination of chemotherapy and immunotherapy to increase the effectiveness of an antitumor immune response is currently regarded as an attractive antitumor strategy. In a pilot clinical trial, we have recently documented an increase of melanoma antigen A (Melan-A)-specific, tumor-reactive, long-lasting effector-memory CD8(+) T cells after the administration of dacarbazine (DTIC) 1 day before peptide vaccination in melanoma patients. Global transcriptional analysis revealed a DTIC-induced activation of genes involved in the immune response and leukocyte activation. To identify the possible mechanisms underlying this improved immune response, we have compared the endogenous and the treatment-induced anti-Melan-A response at the clonal level in patients treated with the vaccine alone or with DTIC plus vaccine. We report a progressive widening of T-cell receptor (TCR) repertoire diversity, accompanied by high avidity and tumor reactivity, only in Melan-A-specific T-cell clones of patients treated with chemoimmunotherapy, with a trend toward longer survival. Differently, patients treated with vaccine alone showed a tendency to narrowing the TCR repertoire diversity, accompanied by a decrease of tumor lytic activity in one patient. Collectively, our findings indicate that DTIC plus vaccination shapes the TCR repertoire in terms of diversity and antitumor response, suggesting that this combined therapy could be effective in preventing melanoma relapse.