Tumor Microenvironment Proteomics: Lessons From Multiple Myeloma.

Although the "seed and soil" hypothesis was proposed by Stephen Paget at the end of the 19th century, where he postulated that tumor cells (seeds) need a propitious medium (soil) to be able to establish metastases, only recently the tumor microenvironment started to be more studied in the field of Oncology. Multiple myeloma (MM), a malignancy of plasma cells, can be considered one of the types of cancers where there is more evidence in the literature of the central role that the bone marrow (BM) microenvironment plays, contributing to proliferation, survival, migration, and drug resistance of tumor cells. Despite all advances in the therapeutic arsenal for MM treatment in the last years, the disease remains incurable. Thus, studies aiming a better understanding of the pathophysiology of the disease, as well as searching for new therapeutic targets are necessary and welcome. Therefore, the present study aimed to evaluate the protein expression profiling of mononuclear cells derived from BM of MM patients in comparison with these same cell types derived from healthy individuals, in order to fill this gap in MM treatment. Proteomic analysis was performed using the mass spectrometry technique and further analyses were done using bioinformatics tools, to identify dysregulated biological pathways and/or processes in the BM microenvironment of patients with MM as a result of the disease. Among the pathways identified in this study, we can highlight an upregulation of proteins related to protein biosynthesis, especially chaperone proteins, in patients with MM. Additionally, we also found an upregulation of several proteins involved in energy metabolism, which is one of the cancer hallmarks. Finally, with regard to the downregulated proteins, we can highlight mainly those involved in different pathways of the immune response, corroborating the data that has demonstrated that the immune system of MM is impaired and, therefore, the immunotherapies that have been studied recently for the treatment of the disease are extremely necessary in the search for a control and a cure for these patients who live with the disease.

Sequential combination of bortezomib and WEE1 inhibitor, MK-1775, induced apoptosis in multiple myeloma cell lines.

INTRODUCTION: Multiple myeloma (MM) remains incurable due to high rates of relapse after various treatment regimens. WEE1 is a cell cycle related gene that regulates the G2/M checkpoint and promotes cell cycle suspension for consequent DNA repair. To date, there are clinical studies for the evaluation of WEE1 inhibitors in the treatment of solid tumors and studies on cell lines of non-MM hematological tumors. OBJECTIVES: To perform in vitro functional studies to verify the effect of the inhibition of WEE1 on MM cell lines viability and its potential as therapeutic target. MATERIAL AND METHODS: WEE1 expression was evaluated in 22 newly diagnosed MM patients and in four MM cell lines, RPMI-8226, U266 and SKO-007 and SK-MM2, by quantitative real-time PCR (qPCR). After treatment with the WEE1 inhibitor (MK-1775), with or without proteasome inhibitor (bortezomib) pretreatment, we assessed cell viability through Prestoblue functional test, microspheres formation in soft agar, and induction of apoptosis and cell cycle alterations by flow cytometry. RESULTS: All MM cell lines showed WEE1 expression by qPCR. RPMI-8226 and U266 showed a 50% reduction in cell viability after 24 h of incubation with MK-1775, at concentrations of 5 µM and 20 µM, respectively. SKO-007 showed dose and time dependence to this drug. Combination therapy with bortezomib and MK-1775 abolished the formation of soft agar microspheres in the RPMI-8226 cell line (also responsive to the use of both drugs) and U266, but SKO-007 was resistant to all drugs, isolated and combined. However, treatment of bortezomib followed by MK-1775 (sequential treatment) versus bortezomib alone showed statistically significant impact on cell lines total apoptosis: 88.8% vs 74.1% in RPMI-8222 (confirmed by cell cycle experiments); 92.5% vs 86.6% in U266; and 60.2% 30.9% on SKO-007 (p < 0.05). CONCLUSION: The sequential combination of bortezomib and WEE1 inhibitor, MK-1775, induced apoptosis in RPMI-8226, U266, and especially SKO-007 cell lines, more efficiently than the use of the same isolated drugs, highlighting its effect in inhibition of proliferation of tumor cells in MM cell lines. Our data suggest that WEE1 can figure as a MM target and that the sequential combination of bortezomib and MK-1775 may be explored in future clinical trials.

Transcriptome Analysis of Mesenchymal Stem Cells from Multiple Myeloma Patients Reveals Downregulation of Genes Involved in Cell Cycle Progression, Immune Response, and Bone Metabolism.

A growing body of evidence suggests a key role of tumor microenvironment, especially for bone marrow mesenchymal stem cells (MSC), in the maintenance and progression of multiple myeloma (MM), through direct and indirect interactions with tumor plasma cells. Thus, this study aimed to investigate the gene expression and functional alterations of MSC from MM patients (MM-MSC) in comparison with their normal counterparts from normal donors (ND-MSC). Gene expression analysis (Affymetrix) was performed in MM-MSC and ND-MSC after in vitro expansion. To validate these findings, some genes were selected to be evaluated by quantitative real time PCR (RT-qPCR), and also functional in vitro analyses were performed. We demonstrated that MM-MSC have a distinct gene expression profile than ND-MSC, with 485 differentially expressed genes (DEG) - 280 upregulated and 205 downregulated. Bioinformatics analyses revealed that the main enriched functions among downregulated DEG were related to cell cycle progression, immune response activation and bone metabolism. Four genes were validated by qPCR - ZNF521 and SEMA3A, which are involved in bone metabolism, and HLA-DRA and CHIRL1, which are implicated in the activation of immune response. Taken together, our results suggest that MM-MSC have constitutive abnormalities that remain present even in the absence of tumors cells. The alterations found in cell cycle progression, immune system activation, and osteoblastogenesis suggest, respectively, that MM-MSC are permanently dependent of tumor cells, might contribute to immune evasion and play an essential role in bone lesions frequently found in MM patients.

Proteasome and heat shock protein 70 (HSP70) inhibitors as therapeutic alternative in multiple myeloma.

HSP70 connects multiple signaling pathways that work synergistically to protect tumor cells from death by proteotoxic stress and represents a possible target to establish a new approach for multiple myeloma treatment. Therefore, bioluminescent cell lines RPMI8226-LUC-PURO and U266-LUC-PURO were treated with HSP70 (VER155008) and/or proteasome (bortezomib) inhibitors and immunodeficient mice were used for subcutaneous xenograft models to evaluate tumor growth reduction and tumor growth inhibition after treatment. Bioluminescence imaging was used to follow tumor response. Treatment with bortezomib showed ∼60% of late apoptosis in RPMI8226-LUC-PURO (without additional benefit of VER155008 in this cell line). However, U266-LUC-PURO showed ∼60% of cell death after treatment with VER155008 (alone or with bortezomib). RPMI8226-LUC-PURO xenograft presented tumor reduction by bioluminescence imaging after treatment with bortezomib, VER155008 or drug combination compared to controls. Treatment with bortezomib, alone or combined with VER155008, showed inhibition of tumor growth assessed by bioluminescence imaging after one week in both RPMI8226-LUC-PURO and U266-LUC-PURO cell lines when compared to controls. In conclusion, our study shows that the combination of proteasome and HSP70 inhibitors induced cell death in tumor cells in vitro (late apoptosis induction) and in vivo (inhibition of tumor growth) with special benefit in U266-LUC-PURO, bearing 17p deletion.

Multiple myeloma cell lines and primary tumors proteoma: protein biosynthesis and immune system as potential therapeutic targets.

Despite great advance in multiple myeloma (MM) treatment since 2000s, it is still an incurable disease and novel therapies are welcome. Therefore, the purpose of this study was to explore MM plasma cells' (MM-PC) proteome, in comparison with their normal counterparts (derived from palatine tonsils of normal donors, ND-PC), in order to find potential therapeutic targets expressed on the surface of these cells. We also aimed to evaluate the proteome of MM cell lines with different genetic alterations, to confirm findings obtained with primary tumor cells. Bone marrow (BM) samples from eight new cases of MM and palatine tonsils from seven unmatched controls were submitted to PC separation and, in addition to two MM cell lines (U266, RPMI-8226), were submitted to protein extraction for mass spectrometry analyses. A total of 81 proteins were differentially expressed between MM-PC and ND-PC - 72 upregulated and nine downregulated; U266 vs. RPMI 8226 cell lines presented 61 differentially expressed proteins - 51 upregulated and 10 downregulated. On primary tumors, bioinformatics analyses highlighted upregulation of protein biosynthesis machinery, as well as downregulation of immune response components, such as MHC class I and II, and complement receptors. We also provided comprehensive information about U266 and RPMI-8226 cell lines' proteome and could confirm some patients' findings.