The mTOR Component, Rictor, Is Regulated By the Microenvironment to Control Dormancy and Proliferative States in Myeloma Cells

Introduction:

Multiple myeloma (MM) cell dormancy and proliferative states, particularly in standard risk patients, are regulated by the BM niches and factors they secrete. Mesenchymal stem cells (MSCs) and their differentiated progeny are key microenvironmental components in MM. We established a reproducible experimental system in which normal MSCs were co-cultured with BM-dependent MM lines for 5 days, at which point MM cells were removed through a trypsinization and replating process (primed MSCs). MSCs cultured alone were similarly processed (unprimed MSCs). Conditioned media (CM) from primed MSCs, but not unprimed MSCs, consistently promoted the growth of primary MM cells from 12 newly diagnosed patients with low-risk MM and 6 patients with high-risk MM (p<0.0003). We hypothesized that primed MSCs represent a high-risk microenvironment akin to growth of MM within focal lesions. The aim of the study was to identify and study microenvironmental pathways that mediate dormancy and proliferative states in MM cells.

Methods:

CD138+ MM cells from 27 newly diagnosed patients were used for growth experiments and molecular characterization. BM-dependent MM lines were established through passaging MM cells from patients with advanced MM in the SCID-rab model.

Unprimed and primed MSCs were molecularly characterized by global gene expression profiling (GEP) and growth factor content in CM was quantified using protein arrays. Proliferation of MSCs was determined by Ki67 immunostaining and cellular senescence by beta-galactosidase (SA-beta-Gal).

MM growth was assessed after culturing primary MM cells with 50% CM from unprimed MSCs, primed MSCs or fresh media for 5 days. Cell survival and proliferation were determined by MTT assay and by detection of annexin V- and Ki67-positive MM cells by flow cytometry. Ultra low depth WGS was performed to assess copy number variation. MM cells were molecularly characterized by GEP, followed by pathway analyses using Ingenuity. Rictor activity was detected in MM cells by Western Blot and double immunostaining for Ki67 and Rictor. Adenoviral-based vectors and siRNA were used for transient RICTOR overexpression and gene expression silencing, respectively.

Results:

Fewer Ki67+ proliferating cells and increased numbers of SA-beta-Gal senescent cells were detected in primed MSCs compared to unprimed MSCs. Primed MSCs expressed a higher level of secreted factors such as CCL5, FGF1, IL6 and IL1B, and reduced expression of FGF7, CXCL12 (SDF1) and IGFBP2 compared to unprimed MSCs. CM of primed MSCs had a higher level of IL6, FGF1 and a lower level of IGFBP2 than CM of unprimed MSCs.

There was no significant difference in the proportion of annexin V+ apoptotic MM cells cultured in CM from unprimed and primed MSCs, whereas the proportion of Ki67+ proliferating cells was 5 fold higher in MM cells treated with primed MSCs CM (p<0.03).

The top genes overexpressed in MM cells treated with primed MSCs CM versus unprimed MSCs CM were related to proliferation, whereas underexpressed genes were related to dormancy including BCL2, RICTOR, and CXCR4. Pathway analyses identified oxidative phosphorylation with mitochondrial dysfunction, cell cycle, mitosis and p53 as the most significantly altered pathways in MM cells treated with primed MSCs CM. WGS revealed similar copy number variation in MM cells treated with unprimed and primed CM, suggesting other mechanisms produced the observed gene expression changes.

mTOR signaling is controlled by major MM growth factors such as IL6 and IGF1, therefore we investigated the role of the mTOR2 component, Rictor, in MM growth. Blocking IL6 or IGF1 with the use of neutralizing antibodies against their receptors inhibited the stimulatory effect of primed MSCs CM on MM cell growth (p<0.01). Western blot analysis and immunostaining revealed a lower level of Rictor in MM cells treated with primed MSCs CM than unprimed MSCs CM. Furthermore, Ki67+ proliferating MM cells were negative for Rictor as assessed by double immunostaining.

Transient overexpression of RICTOR inhibited the growth of 3 MM cell lines by 2-fold (p<0.001), whereas RICTOR siRNA increased the growth of primary MM cells treated with unprimed MSCs CM by 1.8-fold (n=3, p<0.03).

Conclusions:

Primed MSCs possess a senescence phenotype but produce MM growth factors capable of shifting MM cell status from a dormant to proliferative state through downregulation of Rictor expression in MM cells.