Fabricio de Carvalho

TGFβR2 aberrant methylation is a potential prognostic marker and therapeutic target in multiple myeloma

Multiple myeloma (MM) is an incurable hematological malignancy. Different studies demonstrated the occurrence of genetic and epigenetic alterations in MM. The aberrant methylation is one of the most frequent epigenetic alterations in human genome. This study evaluated the aberrant methylation status of 20 genes in 51 MM samples by quantitative methylation‐specific PCR (QMSP) and compared the methylation profile with clinicopathological characteristics of the patients. The QMSP analyses showed that PTGS2 (100.0%), SFN (100.0%), CDKN2B (90.2%), CDH1 (88.2%), ESR1 (72.5%), HIC1 (70.5%), CCND2 (62.7%), DCC (45.1%) and TGFβR2 (39.2%) are frequently hypermethylated in MM while aberrant methylation of RARβ (16.6%), MGMT (12.5%), AIM1 (12.5%), CDKN2A (8.3%), SOCS1 (8.3%), CCNA1 (8.3%) and THBS1 (4.1%) are rare events. There was no methylation of GSTP1, MINT31, p14ARF and RB1 in the samples tested. Hypermethylation of ESR1 was correlated positively with isotype IgA, while aberrant methylation of THBS1 correlated negatively with isotype IgG. Furthermore, hypermethylation of DCC and TGFβR2 were correlated with poor survival. The multivariate analysis showed ISS and TGFβR2 hypermethylation strongly correlated with poor outcome. This study represents the first quantitative evaluation of promoter methylation in MM and our data provide evidence that TGFβR2 hypermethylation, besides ISS, may be useful as prognostic indicator in this disease.

Cancer/Testis Antigen MAGE-C1/CT7: new target for multiple myeloma therapy.

Cancer/Testis Antigens (CTAs) are a promising class of tumor antigens that have a limited expression in somatic tissues (testis, ovary, fetal, and placental cells). Aberrant expression of CTAs in cancer cells may lead to abnormal chromosome segregation and aneuploidy. CTAs are regulated by epigenetic mechanisms (DNA methylation and acetylation of histones) and are attractive targets for immunotherapy in cancer because the gonads are immune privileged organs and anti-CTA immune response can be tumor-specific. Multiple myeloma (MM) is an incurable hematological malignancy, and several CTAs have been detected in many MM cell lines and patients. Among CTAs expressed in MM we must highlight the MAGE-C1/CT7 located on the X chromosome and expressed specificity in the malignant plasma cells. MAGE-C1/CT7 seems to be related to disease progression and functional studies suggests that this CTA might play a role in cell cycle and mainly in survival of malignant plasma cells, protecting myeloma cells against spontaneous as well as drug-induced apoptosis.

Targeting MAGE-C1/CT7 Expression Increases Cell Sensitivity to the Proteasome Inhibitor Bortezomib in Multiple Myeloma Cell Lines

The MAGE-C1/CT7 encodes a cancer/testis antigen (CTA), is located on the chromosomal region Xq26–27 and is highly polymorphic in humans. MAGE-C1/CT7 is frequently expressed in multiple myeloma (MM) that may be a potential target for immunotherapy in this still incurable disease. MAGEC1/CT7 expression is restricted to malignant plasma cells and it has been suggested that MAGE-C1/CT7 might play a pathogenic role in MM; however, the exact function this protein in the pathophysiology of MM is not yet understood. Our objectives were (1) to clarify the role of MAGE-C1/CT7 in the control of cellular proliferation and cell cycle in myeloma and (2) to evaluate the impact of silencing MAGE-C1/CT7 on myeloma cells treated with bortezomib. Myeloma cell line SKO-007 was transduced for stable expression of shRNA-MAGE-C1/CT7. Downregulation of MAGE-C1/CT7 was confirmed by real time quantitative PCR and western blot. Functional assays included cell proliferation, cell invasion, cell cycle analysis and apoptosis. Western blot showed a 70–80% decrease in MAGE-C1/CT7 protein expression in inhibited cells (shRNA-MAGE-C1/CT7) when compared with controls. Functional assays did not indicate a difference in cell proliferation and DNA synthesis when inhibited cells were compared with controls. However, we found a decreased percentage of cells in the G2/M phase of the cell cycle among inhibited cells, but not in the controls (p<0.05). When myeloma cells were treated with bortezomib, we observed a 48% reduction of cells in the G2/M phase among inhibited cells while controls showed 13% (empty vector) and 9% (ineffective shRNA) reduction, respectively (p<0.01). Furthermore, inhibited cells treated with bortezomib showed an increased percentage of apoptotic cells (Annexin V+/PI-) in comparison with bortezomib-treated controls (p<0.001). We found that MAGE-C1/CT7 protects SKO-007 cells against bortezomib-induced apoptosis. Therefore, we could speculate that MAGE-C1/CT7 gene therapy could be a strategy for future therapies in MM, in particular in combination with proteasome inhibitors.

Frequency and prognostic relevance of cancer testis antigen 45 expression in multiple myeloma.

OBJECTIVE This study aims to analyze the expression of cancer testis antigen 45 (CT45) in normal tissues and in plasma cell disorders and to identify possible associations with clinical data and prognosis in multiple myeloma (MM) patients. MATERIALS AND METHODS Expression of CT45 was studied in 20 normal tissues (testis, placenta, skeletal muscle, bladder, lung, spleen, heart, brain and fetal brain, thymus, uterus, stomach, mammary gland, pancreas, prostate, small intestine, kidney, adrenal gland, spinal cord, colon, and one pool of 10 normal bone marrow samples) and bone marrow aspirates from 3 monoclonal gammopathies of undetermined significance, 5 solitary plasmacytomas, 61 newly diagnosed MM patients and MM cell line U266 by reverse transcriptase polymerase chain reaction. RESULTS CT45 was positive in 3 of 20 (15%) normal tissues tested: lung, brain (both fetal and adult), and spinal cord. Among monoclonal gammopathies, CT45 was positive in 2 of 5 (40%) solitary plasmacytomas bone marrow aspirates, 10 of 61 (16%) MM bone marrow aspirates, and in the U266 MM cell line. CONCLUSIONS We did not find associations between bone marrow histology and CT45 expression. However, we demonstrated for the first time that positive expression of CT45 was associated with poor prognostic (International Staging System) and poor outcomes in MM patients, meaning that CT45-positive cases presented seven times more chance of worse evolution than the negative ones.

Number of expressed cancer/testis antigens identifies focal adhesion pathway genes as possible targets for multiple myeloma therapy

Considering that the importance of cancer/testis (CT) antigens in multiple myeloma (MM) biology is still under investigation, the present study aimed to: (1) identify genes differentially expressed in MM using microarray analysis of plasma cell samples, separated according to the number of expressed CTs; (2) examine possible pathways related to MM pathogenesis; (3) validate the expression of candidate genes by quantitative real-time PCR (RQ-PCR). Three samples predominantly positive (>6 expressed), including the U266 cell line, and three samples predominantly negative (0 or 1 expressed CT for the 13 analyzed CT antigens), were submitted for microarray analysis. Validation by RQ-PCR from 24 MM samples showed that the ITGA5 gene was downregulated in predominantly positive (>6 expressed CTs, p = 0.0030) and in tumor versus normal plasma cells (p = 0.0182). The RhoD gene was overexpressed in tumor plasma cells when compared to normal plasma cells (p = 0.0339). Results of the microarray analysis corroborate the hypothesis that MM could be separated into predominantly positive and predominantly negative expression. The differential expression of ITGA5 and RhoD suggests disruption of the focal adhesion pathway in MM and offers a new target field to be explored in this disease.

Analysis of protein profile expressed on the membrane surface of plasma cells, stromal cells and mononuclear cells from bone marrow microenvironment of multiple myeloma patients by mass spectrometry

Background Multiple Myeloma (MM) is a malignant proliferation of B-cells with plasma cell differentiation. Interactions of plasma cells with stromal cells and other cells in bone marrow (BM) microenvironment are responsible for MM progression. Screening of proteins expressed on the membrane surface of plasma cells, stromal cells and mononuclear cells from MM patients by mass spectrometry (MS) may identify new prognostic and therapeutic targets in this incurable disease.