Luca Sigalotti

Endoglin: An accessory component of the TGF-β-binding receptor-complex with diagnostic, prognostic, and bioimmunotherapeutic potential in human malignancies

Endoglin (CD105) is a cell membrane glycoprotein over‐expressed on highly proliferating endothelial cells in culture, and on endothelial cells of angiogenetic blood vessels within benign and malignant tissues. CD105 binds several factors of the Transforming Growth Factor (TGF)‐β superfamily, and its over‐expression modulates cellular responses to TGF‐β1. The complex of experimental findings accumulated in the last few years strongly indicate that CD105 is a powerful marker of angiogenesis, and that it might play a critical role in the pathogenesis of vascular diseases and in tumor progression. In this paper, we will review the structural, biological and functional features of CD105, as well as its distribution within normal and neoplastic tissues, emphasizing its foreseeable role as a molecular target for new diagnostic and bioimmunotherapeutic approaches in human malignancies.

Intratumor Heterogeneity of Cancer/Testis Antigens Expression in Human Cutaneous Melanoma Is Methylation-Regulated and Functionally Reverted by 5-Aza-2′-deoxycytidine

Cancer/testis antigens (CTA) are suitable targets for immunotherapy of human malignancies, and clinical trials are mainly focusing on MAGE-A3. However, the heterogeneous intratumor expression of CTA may hamper the effectiveness of CTA-directed vaccination through the emergence of CTA-negative neoplastic clones. We investigated the intratumor heterogeneity of CTA in human melanoma and the underlying molecular mechanism(s) at clonal level using 14 single cell clones generated from the melanoma lesion Mel 313. Reverse transcription-PCR revealed a highly heterogeneous expression of MAGE-A1, -A2, -A3, -A4, -A6, GAGE 1–6, SSX 1–5, and PRAME among melanoma clones. Only nine clones expressed MAGE-A3 and competitive reverse transcription-PCR identified relative differences in the number of mRNA molecules of up to 130-fold between clones 5 and 14. This clonal heterogeneity of MAGE-A3 expression correlated with the methylation status of specific CpG dinucleotides in MAGE-A3 promoter: i.e., hypomethylated CpG dinucleotides at positions −321, −151, −19, −16, −5, −2, +21, and +42 were found in clones expressing high but not low levels of MAGE-A3. Supporting the role of DNA methylation in generating the intratumor heterogeneity of CTA, the DNA hypomethylating agent 5-aza-2′-deoxycytidine (5-AZA-dCyd) invariably induced their expression in all CTA-negative clones. Furthermore, 5-AZA-dCyd–treatment reduced to 6 folds the differential expression of MAGE-A3 between clones 5 and 14, which became recognized to a similar extent by T cells specific for a MAGE-A–encoded peptide. These findings identify promoter methylation as directly responsible for the intratumoral heterogeneity of therapeutic CTA in melanoma and foresee the use of 5-AZA-dCyd to overcome the limitations set by their intratumor heterogeneous expression to CTA-based vaccine therapy.

The biology of cancer testis antigens: Putative function, regulation and therapeutic potential

Cancer testis antigens (CTA) are a large family of tumor‐associated antigens expressed in human tumors of different histological origin, but not in normal tissues except for testis and placenta. This tumor‐restricted pattern of expression, together with their strong in vivo immunogenicity, identified CTA as ideal targets for tumor‐specific immunotherapeutic approaches, and prompted the development of several clinical trials of CTA‐based vaccine therapy. Driven by this practical clinical interest, a more detailed characterization of CTA biology has been recently undertaken. So far, at least 70 families of CTA, globally accounting for about 140 members, have been identified. Most of these CTA are expressed during spermatogenesis, but their function is still largely unknown. Epigenetic events, particularly DNA methylation, appear to be the primary mechanism regulating CTA expression in both normal and transformed cells, as well as in cancer stem cells. In view of the growing interest in CTA biology, the aim of this review is to provide the most recent information on their expression, regulation and function, together with a brief summary of the major clinical trials involving CTA as therapeutic agents. The pharmacologic modulation of CTA expression profiles on neoplastic cells by DNA hypomethylating drugs will also be discussed as a feasible approach to design new combination therapies potentially able to improve the clinical efficacy of currently adopted CTA‐based immunotherapeutic regimens in cancer patients.

Epigenetic Drugs as Pleiotropic Agents in Cancer Treatment: Biomolecular Aspects and Clinical Applications

In the last three decades huge efforts have been made to characterize genetic defects responsible for cancer development and progression, leading to the comprehensive identification of distinct cellular pathways affected by the alteration of specific genes. Despite the undoubtable role of genetic mechanisms in triggering neoplastic cell transformation, epigenetic modifications (i.e., heritable changes of gene expression that do not derive from alterations of the nucleotide sequence of DNA) are rapidly emerging as frequent alterations that often occur in the early phases of tumorigenesis and that play an important role in tumor development and progression. Epigenetic alterations, such as modifications in DNA methylation patterns and post‐translational modifications of histone tails, behave extremely different from genetic modifications, being readily revertable by “epigenetic drugs” such as inhibitors of DNA methyl transferases and inhibitors of histone deacetylases. Since epigenetic alterations in cancer cells affect virtually all cellular pathways that have been associated to tumorigenesis, it is not surprising that epigenetic drugs display pleiotropic activities, being able to concomitantly restore the defective expression of genes involved in cell cycle control, apoptosis, cell signaling, tumor cell invasion and metastasis, angiogenesis and immune recognition. Prompted by this emerging clinical relevance of epigenetic drugs, this review will focus on the large amount of available data, deriving both from in vitro experimentations and in vivo pre‐clinical and clinical studies, which clearly indicate epigenetic drugs as effective modifiers of cancer phenotype and as positive regulators of tumor cell biology with a relevant therapeutic potential in cancer patients. J. Cell. Physiol. 212: 330–344, 2007.

CXCR6, a Newly Defined Biomarker of Tissue-Specific Stem Cell Asymmetric Self-Renewal, Identifies More Aggressive Human Melanoma Cancer Stem Cells

Background A fundamental problem in cancer research is identifying the cell type that is capable of sustaining neoplastic growth and its origin from normal tissue cells. Recent investigations of a variety of tumor types have shown that phenotypically identifiable and isolable subfractions of cells possess the tumor-forming ability. In the present paper, using two lineage-related human melanoma cell lines, primary melanoma line IGR39 and its metastatic derivative line IGR37, two main observations are reported. The first one is the first phenotypic evidence to support the origin of melanoma cancer stem cells (CSCs) from mutated tissue-specific stem cells; and the second one is the identification of a more aggressive subpopulation of CSCs in melanoma that are CXCR6+. Methods/Findings We defined CXCR6 as a new biomarker for tissue-specific stem cell asymmetric self-renewal. Thus, the relationship between melanoma formation and ABCG2 and CXCR6 expression was investigated. Consistent with their non-metastatic character, unsorted IGR39 cells formed significantly smaller tumors than unsorted IGR37 cells. In addition, ABCG2+ cells produced tumors that had a 2-fold greater mass than tumors produced by unsorted cells or ABCG2- cells. CXCR6+ cells produced more aggressive tumors. CXCR6 identifies a more discrete subpopulation of cultured human melanoma cells with a more aggressive MCSC phenotype than cells selected on the basis of the ABCG2+ phenotype alone. Conclusions/Significance The association of a more aggressive tumor phenotype with asymmetric self-renewal phenotype reveals a previously unrecognized aspect of tumor cell physiology. Namely, the retention of some tissue-specific stem cell attributes, like the ability to asymmetrically self-renew, impacts the natural history of human tumor development. Knowledge of this new aspect of tumor development and progression may provide new targets for cancer prevention and treatment.

Epigenetics of human cutaneous melanoma: setting the stage for new therapeutic strategies

Cutaneous melanoma is a very aggressive neoplasia of melanocytic origin with constantly growing incidence and mortality rates world-wide. Epigenetic modifications (i.e., alterations of genomic DNA methylation patterns, of post-translational modifications of histones, and of microRNA profiles) have been recently identified as playing an important role in melanoma development and progression by affecting key cellular pathways such as cell cycle regulation, cell signalling, differentiation, DNA repair, apoptosis, invasion and immune recognition. In this scenario, pharmacologic inhibition of DNA methyltransferases and/or of histone deacetylases were demonstrated to efficiently restore the expression of aberrantly-silenced genes, thus re-establishing pathway functions. In light of the pleiotropic activities of epigenetic drugs, their use alone or in combination therapies is being strongly suggested, and a particular clinical benefit might be expected from their synergistic activities with chemo-, radio-, and immuno-therapeutic approaches in melanoma patients. On this path, an important improvement would possibly derive from the development of new generation epigenetic drugs characterized by much reduced systemic toxicities, higher bioavailability, and more specific epigenetic effects.

Functional Up-regulation of Human Leukocyte Antigen Class I Antigens Expression by 5-aza-2′-deoxycytidine in Cutaneous Melanoma: Immunotherapeutic Implications

Purpose: To investigate the potential of the DNA hypomethylating agent 5-aza-2′-deoxycytidine (5-aza-CdR) to improve the effectiveness of immunotherapeutic approaches against melanocyte differentiation antigens. Experimental Design: The effect of 5-aza-CdR on the constitutive expression of gp100 was investigated in 11 human melanoma cell lines by real-time reverse transcription-PCR and indirect immunofluorescence (IIF) analyses. 5-aza-CdR–mediated changes in the levels of expression of human leukocyte antigen (HLA) class I antigens and HLA-A2 allospecificity, intercellular adhesion molecule-1 (ICAM-1), and leukocyte-function–associated antigen-3 were investigated by IIF analysis on melanoma cells under study. The recognition of gp100-positive Mel 275 melanoma cells, treated or not with 5-aza-CdR, by HLA-A2–restricted gp100(209–217)-specific CTL was investigated by 51Cr-release assays, IFN-γ release and IFN-γ ELISPOT assays. Results: The constitutive expression of gp100 was not affected by 5-aza-CdR on all melanoma cells investigated. Compared with untreated cells, the exposure of Mel 275 melanoma cells to 5-aza-CdR significantly (P < 0.05) up-regulated their expression of HLA class I antigens and of ICAM-1. These phenotypic changes significantly (P < 0.05) increased the lysis of 5-aza-CdR–treated Mel 275 melanoma cells by gp100-specific CTL and increased their IFN-γ release. 5-aza-CdR treatment of Mel 275 cells also induced a higher number of gp100-specific CTL to secrete IFN-γ. Conclusions: Treatment with 5-aza-CdR improves the recognition of melanoma cells by gp100-specific CTL through the up-regulation of HLA class I antigens expression; ICAM-1 also contributes to this phenomenon. These findings highlight a broader range of therapeutic implications of 5-aza-CdR when used in association with active or adoptive immunotherapeutic approaches against a variety of melanoma-associated antigens.

Epigenetic targets for immune intervention in human malignancies

Emerging evidences suggest that epigenetic events associated with tumor development and progression, such as deregulated methylation of CpG dinucleotides and aberrant histone acetylation, may impair the immunogenic potential of cancer cells. In fact, DNA hypermethylation and/or histone deacetylation contribute to the absent or downregulated expression of different components of the ‘tumor recognition complex’ (i.e., HLA class I antigens, cancer/testis antigens and accessory/costimulatory molecules) in solid and hemopoietic human malignancies. However, pharmacologic agents that induce DNA hypomethylation or inhibit histone deacetylation can modify these epigenetic phenomena, restoring the defective expression of selected components of the ‘tumor recognition complex’ in cancer cells. These antigenic modifications positively modulate the immunogenicity and the immune recognition of cancer cells, making epigenetic drugs attractive agents to design new combined chemoimmunotherapeutic strategies for the treatment of cancer patients.

Cancer testis antigens in human melanoma stem cells: expression, distribution, and methylation status.

Neoplastic populations with stem cell potential have been most recently identified in human cutaneous melanoma, and initially characterized for their phenotypic profile. Being melanoma stem cells (MSC) the most desirable target of therapeutic intervention, we asked whether they express the epigenetically‐regulated cancer testis antigens (CTA) on which melanoma immunotherapy is increasingly focusing. Reverse transcription‐PCR analyses identified the presence of the large majority of investigated CTA (i.e., MAGE, GAGE, NY‐ESO, and SSX families) in different MSC populations. MSC expressed MAGE‐A proteins as detected by western blot; noteworthy, the distribution of MAGE‐A proteins was highly homogeneous within given MSC populations as shown by confocal immunofluorescence. Promoter methylation studies unveiled a homogeneously‐demethylated MAGE‐A3 promoter that paired MAGE‐A3 expression in MSC. Altogether these findings demonstrate that MSC can be efficiently targeted by CTA‐directed immunotherapeutic approaches, and suggest that epigenetic patterns most likely drive the expression of CTA in MSC as previously shown for melanoma cells. J. Cell. Physiol. 215: 287–291, 2008.

Clinical Studies With Anti–CTLA-4 Antibodies in Non-melanoma Indications

Available medical treatments have limited impact on the survival of patients with advanced cancer; therefore, new therapeutic strategies able to generate more effective hosts immune responses against neoplastic cells are being actively pursued. Among these, a recent approach involves targeting of cytotoxic T-lymphocyte antigen-4 (CTLA-4), a key immune checkpoint molecule, by monoclonal antibodies (mAbs). Ipilimumab and tremelimumab represent the prototypes of this new class of immunomodulating mAb and have been extensively tested in metastatic melanoma with highly promising results. The clinical activity observed in melanoma has served as a model to exploit the therapeutic potential of CTLA-4 blockade in a variety of human malignancies. Along this line, early-phase trials with anti-CTLA-4 mAbs have been completed or are ongoing in tumors of different histotype. Results are demonstrating the feasibility, safety, and activity of these agents, thus suggesting a promising therapeutic role to be further investigated in phase II/III trials in a wide range of tumors. This review summarizes the main trials with ipilimumab and tremelimumab in tumors of different histotypes, excluding cutaneous melanoma, which is extensively described in other chapters of this issue of Seminars in Oncology.