Sandra Coral

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.

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.

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.

Methylation levels of the "long interspersed nucleotide element-1" repetitive sequences predict survival of melanoma patients

BackgroundThe prognosis of cutaneous melanoma (CM) differs for patients with identical clinico-pathological stage, and no molecular markers discriminating the prognosis of stage III individuals have been established. Genome-wide alterations in DNA methylation are a common event in cancer. This study aimed to define the prognostic value of genomic DNA methylation levels in stage III CM patients.MethodsOverall level of genomic DNA methylation was measured using bisulfite pyrosequencing at three CpG sites (CpG1, CpG2, CpG3) of the Long Interspersed Nucleotide Element-1 (LINE-1) sequences in short-term CM cultures from 42 stage IIIC patients. The impact of LINE-1 methylation on overall survival (OS) was assessed using Cox regression and Kaplan-Meier analysis.ResultsHypomethylation (i.e., methylation below median) at CpG2 and CpG3 sites significantly associated with improved prognosis of CM, CpG3 showing the strongest association. Patients with hypomethylated CpG3 had increased OS (P = 0.01, log-rank = 6.39) by Kaplan-Meyer analysis. Median OS of patients with hypomethylated or hypermethylated CpG3 were 31.9 and 11.5 months, respectively. The 5 year OS for patients with hypomethylated CpG3 was 48% compared to 7% for patients with hypermethylated sequences. Among the variables examined by Cox regression analysis, LINE-1 methylation at CpG2 and CpG3 was the only predictor of OS (Hazard Ratio = 2.63, for hypermethylated CpG3; 95% Confidence Interval: 1.21-5.69; P = 0.01).ConclusionLINE-1 methylation is identified as a molecular marker of prognosis for CM patients in stage IIIC. Evaluation of LINE-1 promises to represent a key tool for driving the most appropriate clinical management of stage III CM patients.

Melanoma cells constitutively release an anchor-positive soluble form of protectin (sCD59) that retains functional activities in homologous complement-mediated cytotoxicity.

Protectin (CD59), a glycosylphosphatidylinositol-anchored cell membrane glycoprotein, is differentially expressed on melanocytic cells and represents the main restriction factor of C-mediated lysis of melanoma cells. In this study, we report that CD59-positive melanoma cells constitutively release a soluble form of CD59 (sCD59), and that its levels directly correlate (r = 0.926; P < 0.05) with the amount of membrane-bound CD59. SDS-PAGE analysis showed that the molecular components of sCD59 are similar to those of cellular CD59 expressed by melanoma cells. Melanoma-released sCD59 is anchor positive since it inserts into cell membranes of homologous cells that transiently increase their expression of CD59. Moreover, sCD59 is functional: it blocks the binding of the anti-CD59 mAb YTH53.1 to melanoma cells and reverses its effects on C-mediated lysis. In fact, preincubation of mAb YTH53.1 with scalar doses of conditioned media of CD59-positive but not of CD59-negative melanoma cells reduced significantly (P < 0.05), and in a dose-dependent fashion, the enhancement of C-mediated lysis of anti-GD3-sensitized melanoma cells induced by the masking of cellular CD59 by mAb YTH53.1. Altogether, these data demonstrate that CD59-positive human melanoma cells release a soluble form of CD59 that is structurally similar to cellular CD59, retains its anchoring ability, is functional, and may impair the effectiveness of clinical approaches to humoral immunotherapy for human melanoma.

Phenotypic and functional changes of human melanoma xenografts induced by DNA hypomethylation: Immunotherapeutic implications

Emerging in vitro evidence points to an immunomodulatory activity of DNA hypomethylating drugs in human malignancies. We investigated the potential of 5‐aza‐2′‐deoxycytidine (5‐AZA‐CdR) to modulate the expression of cancer testis antigens (CTA) and of HLA class I antigens by melanoma xenografts, and the resulting modifications in immunogenicity of neoplastic cells. Three primary cultures of melanoma cells, selected for immune phenotype and growth rate, were grafted into BALB/c nu/nu mice that were injected intraperitoneally with different dose‐ and time‐schedules of 5‐AZA‐CdR. Molecular analyses demonstrated a de novo long‐lasting expression of the CTA MAGE‐1, ‐2, ‐3, ‐4, ‐10, GAGE 1–6, NY‐ESO‐1, and the upregulation of MAGE‐1, MAGE‐3, and NY‐ESO‐1 levels in melanoma xenografts from 5‐AZA‐CdR‐treated mice. Serological and biochemical analyses identified a de novo expression of NY‐ESO‐1 protein and a concomitant and persistent upregulation of HLA class I antigens and of HLA‐A1 and ‐A2 alleles. Immunization of BALB/c mice with 5‐AZA‐CdR‐treated melanoma cells generated high titer circulating anti‐NY‐ESO‐1 antibodies. Altogether, the data obtained identify an immunomodulatory activity of 5‐AZA‐CdR in vivo and strongly suggest for its clinical use to design novel strategies of CTA‐based chemo‐immunotherapy for melanoma patients. J. Cell. Physiol. 207: 58–66, 2006.

Methylation-regulated expression of HLA class I antigens in melanoma.

Dear Sir, Serrano et al. recently reported that prolonged treatment with high doses of the DNA hypomethylating agent 5-AZA2 -deoxycitydine (5-AZA-CdR) induced de novo expression of the mRNA encoded by HLA-A, -B and -C loci in the HLA class I-negative melanoma cell line MSR3-mel.1 Combined treatment with 5-AZA-CdR and interferon (IFN)resulted in the expression of HLA-A and -B proteins on the cell membrane of MSR3-mel melanoma cells and induced higher levels of HLA-B antigens. Furthermore, the authors demonstrated that exposure of MSR3-mel melanoma cells to 5-AZA-CdR, IFNand tumor necrosis (TNF)resulted in their recognition by HLA-A and -B restricted tumor-specific cytotoxic T cells (CTL). In light of these findings, the authors suggested that DNA hypermethylation is a new molecular mechanism generating complete loss of HLA class I antigens expression in human melanomas and concluded that pharmacologic modulation of DNA methylation represents a possible intervention for cancer treatment, which, however, is limited by the high toxicity of available DNA hypomethylating drugs. We have shown that 5-AZA-CdR significantly (p 0.05) and persistently upregulates the constitutive expression of HLA class I antigens and of HLA-A1 and -A2 allospecificities on primary cultures of metastatic melanoma cells.2 These findings, and the concomitant de novo expression or upregulation of Cancer Testis Antigens (CTA) by 5-AZA-CdR,2,3 led us to propose for its perspective clinical usefulness to implement new therapeutic strategies in melanoma patients.2,3 Nevertheless, we found that 5-AZA-CdR did not induce de novo expression of the HLA-A2 allospecificity on HLA class I antigens-positive but HLA-A2-negative melanoma cultures (data not shown). These evidences suggested to us that DNA hypermethylation does not represent a mechanism utilized by melanoma cells to turn off their expression of HLA class I antigens. The data generated with MSR3-mel melanoma cells,1 which clearly point to a different conclusion, prompted us to extend our preliminary findings. To this end, primary cultures of metastatic melanoma cells from patients with no history of chemotherapy or immunotherapy, 4 of which showed complete loss of HLA class I antigens and 4 with selective loss of the HLA-A2 allospecificity (Table I), were pulsed 4 times with 1 M 5-AZA-CdR every 12 hr and further cultured as previously described.2,3 5-AZA-CdR constantly failed to induce de novo expression of HLA class I antigens and of the HLA-A2 allospecificity on investigated melanoma cells (Table I); however, as expected, it upregulated their baseline expression (Table I). The DNA hypomethylating effect of 5-AZA-CdR was confirmed by the induction or by the upregulation of the expression of the CTA MAGE-1, -2, -3, GAGE 1-2, NYESO-1 and SSX 1–5 in melanoma cultures studied and by a significant inhibition of cellular replication (data not shown). In spite of these experimental evidences, we reasoned that higher concentrations of 5-AZA-CdR and/or longer exposure to the demethylating agent could restore the deficient expression of HLA class I molecules. However, in our present and previous experience with more than 40 different melanoma cultures, we were invariably unable to increase the concentrations of 5-AZA-CdR and/or to prolong the length of treatment over 4 days, as this resulted in complete destruction of neoplastic cells. Therefore, according to the culture conditions described by Serrano et al., MSR3-mel likely represents a melanoma cell line with an exquisite resistance to the growth inhibitory and cytotoxic activity of 5-AZA-CdR.4 We have also shown that opposite to IFN, treatment with 5-AZA-CdR upregulates the expression of HLA-A and -B antigens to a similar extent on melanoma cells.5 Thus, it will be interesting to dissect in depth the relative contribution of 5-AZA-CdR, IFNand/or TNFin the reported recognition of MSR3-mel melanoma cells by effector CTL. In fact, the absolute requirement of all 3 therapeutic components to achieve the observed functional results would add great complexity to a foreseeable therapeutic schema, while further increasing the toxicity of DNA hypomethylating agents used alone. Altogether, the experimental evidences available strongly suggest that DNA hypermethylation is a molecular mechanism that melanoma cells consistently utilize to downregulate but not to switch off their expression of HLA class I antigens; in this respect, MSR3-mel melanoma cells very likely represent the exception rather than the rule. Furthermore, we would