MicroRNA-92 Expression in CD133+ Melanoma Stem Cells Regulates Immunosuppression in the Tumor Microenvironment via Integrin-Dependent Activation of TGFβ

Abstract

In addition to being refractory to treatment, melanoma cancer stem cells (CSC) are known to suppress host antitumor immunity, the underlying mechanisms of which need further elucidation. In this study, we established a novel role for miR-92 and its associated gene networks in immunosuppression. CSCs were isolated from the B16-F10 murine melanoma cell line based on expression of the putative CSC marker CD133 (Prominin-1). CD133⁺ cells were functionally distinct from CD133^- cells and showed increased proliferation in vitro and enhanced tumorigenesis in vivo. CD133⁺ CSCs also exhibited a greater capacity to recruit immunosuppressive cell types during tumor formation, including FoxP3⁺ Tregs, myeloid-derived suppressor cells (MDSC), and M2 macrophages. Using microarray technology, we identified several miRs that were significantly downregulated in CD133⁺ cells compared with CD133^- cells, including miR-92. Decreased expression of miR-92 in CSCs led to higher expression of target molecules integrin α_V and α₅ subunits, which, in turn, enhanced TGFβ activation, as evidenced by increased phosphorylation of SMAD2. CD133⁺ cells transfected with miR-92a mimic and injected in vivo showed significantly decreased tumor burden, which was associated with reduced immunosuppressive phenotype intratumorally. Using The Cancer Genome Atlas database of patients with melanoma, we also noted a positive correlation between integrin α₅ and TGFβ1 expression levels and an inverse association between miR-92 expression and integrin alpha subunit expression. Collectively, this study suggests that a miR-92-driven signaling axis involving integrin activation of TGFβ in CSCs promotes enhanced tumorigenesis through induction of intratumoral immunosuppression. SIGNIFICANCE: CD133⁺ cells play an active role in suppressing melanoma antitumor immunity by modulating miR-92, which increases influx of immunosuppressive cells and TGFβ1 expression.

Figure 1:. CSCs are enriched in the CD133⁺ population and are functionally distinct from their CD133⁻ counterparts.

CD133⁺ cells form palpable tumors and display elevated growth kinetics in a syngeneic mouse model (A,B) (n = 6 per group). Tumor volumes represent mean tumor volume ± SEM. Mice (4/6) injected with CD133⁻ cells formed tumors while all (6/6) mice injected with CD133⁺ cells formed tumors. In vitro colony formation (C) and non-adherent oncosphere formation (D) was significantly increased in CD133-expressing populations. Images depict anchorage-dependent colony growth (C) and anchorage-independent oncosphere growth in SFM media (D) and are representative of data collected from three independent experiments. Statistical significance was determined at p < 0.05 and was denoted by an asterisk (*). P-values have been provided where appropriate. Dissociation, labeling, and analysis of representative tumor samples initiated by CD133⁺ and CD133⁻ B16-F10 melanoma cells demonstrated a significant shift in lymphocyte (E), MDSC (F), and macrophage (G) populations. Statistical analysis on samples generated from CD133⁺ and CD133⁻ initiated tumors identified several significant changes associated with each group (H). SPADE analysis further demonstrated the alterations in immune cell infiltration of the TME between CD133⁺ cells and CD133⁻ cells (I). Flow plots and SPADE analysis were generated from representative data collected from two independent in vivo experiments. Significance was determined by Student’s t test (p < 0.05) and is denoted by an asterisk. (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 2:. Microarray analysis of CD133⁺ B16-F10 cells compared to CD133⁻ cells.

Analysis of microarray data demonstrated significant disparities in miR expression between CSC and non-CSC compartments outlined in blue (CD133+ = orange, CD133- = green); all miRNA are shown in the above heatmap and sorted by fold-change (CD133-positive relative to CD133-negative in ascending order). Several of these miRNAs were validated using qRT-PCR (A). miR-92 was identified to target several cancer-associated gene networks using Metacore (B) and Ingenuity (C) pathway analysis tools. Using sequence alignment software, miR-92 is highly predicted to target mRNAs for integrin alpha subunits involved in activation of secreted TGF-β (D).

Figure 3:. RNA and protein expression analysis of integrin subunits and TGF-β associated signaling molecules.

Total RNA was isolated from CD133⁺ and CD133⁻ cells and assessed for integrin subunit expression (A, top) and TGF-β signaling through SMAD2 (A, bottom) by qRT-PCR. mRNA expression levels were normalized to the CD133⁻ cell phenotype. Protein level expression for integrin α_v and α₅ subunits as well as SMAD2 phosphorylation was assessed by western blot. Actin and GAPDH were used as reference proteins for western blots assessing integrin subunit expression and SMAD signaling, respectively. Band intensities were calculated using ImageJ software and relative densitometric intensity (normalized to Actin/GAPDH) are displayed below the appropriate band. PCR and immunblot analysis data were gathered from two independent experiments in which samples were run in triplicate (qRT-PCR) or in duplicate (immunoblot). Significance was determined by Student’s t test (p < 0.05) and is denoted by an asterisk. (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 4 :. Experimental metastasis model of murine melanoma using CD133-sorted cell populations.

CD133-sorted B16-F10 cells were injected i.v. into C57Bl/6 mice and allowed to colonize the pulmonary tissues (A). Pulmonary lesions were counted and measured upon resection of lung tissues and plotted as mean number of metastases ± SEM. CD133⁺ cells formed larger and more abundant macrometastases when compared to CD133⁻ cells (A). Lungs from tumor bearing mice were dissociated and labeled using myeloid (B, top) and lymphocyte (B, bottom) panels to identify shifts in subsets of T cells, macrophages, and MDSCs from the TME generated by each tumor phenotype. Representative flow plots from lung tissues analyzed by myeloid (C) and lymphocyte (D) panels demonstrated the significant shifts in immune cell phenotypes. Downstream analysis using SPADE software depicted changes in the TME between tissues colonized by CD133⁺ and CD133⁻ melanoma cells (E). Legend provided is based on these analyses (E, gray scale). The panels identified significant shifts in immune cell phenotypes in both lymphocyte (E, top) and myeloid (E, bottom) panels. Experimental metastasis models were repeated once (n = 5 per group). Statistical significance was determined by Student’s t-test and was denoted by an asterisk. P-values are provided where appropriate.

Figure 5:. ELISA for free/active TGF-β in a co-culture model demonstrated the enhanced ability of CSCs to convert secreted (inactive) TGF-β to its active form.

FACS-sorted CD133 –positive and –negative populations were co-cultured with splenocytes from C57Bl/6 mice for 8h (A,top) and 24h (A,bottom) at a 1:1 E:T ratio, and the resulting supernatants were analyzed for activated TGF-β. Flow cytometry and subsequent SPADE analysis was conducted on the resulting splenocytes to identify shifts in cell phenotypes after 24h co-culture (B-D). A legend for SPADE analysis has been provided (B). Flow cytometry panels resulting from co-cultures using both CD133⁺ (C) and CD133⁻ (D) are also provided. ELISA and flow cytometric analysis were repeated twice as independent experiments. Statistical significance was determined by Student’s t-test (p < 0.05) and is denoted by an asterisk. (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 6 :. miR-92a regulated the integrin-TGF-β axis and inhibited tumor growth in vivo.

B16-F10 cells sorted on positive CD133 expression were transfected with miR-92a mimic, inhibitor, and a combination of mimic and inhibitor for 48 hours. Gene expression was measured by qRT-PCR for miR-92a, ITGAV, ITGA5, TGFB1, and SMAD2 following the transfection and isolation of total RNA; all groups were normalized to mock transfected cells as a reference (A). CD133+ cells isolated from the B16 cell line were transfected with miR-92 mimic or lipid. Transfected cells were injected s.c. into C57bl/6 mice and allowed to form tumors over 14 days (B). Tumor bearing mice were sacrificed upon endpoint and tumors were dissociated, labeled with panels of antibodies against phenotypic markers for lymphocytes and monocytes, and analyzed by flow cytometry (C-F) as previously described. Statistical analyses were performed using a Student’s t-test and one way ANOVA with significance determined at p < 0.05. Statistical significance is denoted in Panel A as follows: a = significant from 2,3,4; b = significant from 1,3,4; c = significant from 1,2,4; d = significant from 1,2,3; e = significant from 2,3. Statistical significance in Panel F is denoted as follows: *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 7:. TCGA validates the association between integrin α5 and TGF-β and miR-92 in human clinical samples of melanoma.

Using cBioPortal and UCSC Xena, we probed datasets obtained from patients with skin cancer in order to identify correlations between mRNA level expression of integrin α5 and TGF-β1 (A). We identified several clinical specimens in which high expression of TGF-β was associated with elevated expression of integrin α5 (A) as highlighted by the rectangles (red = high expression, blue = low expression). Using the Pearson and Spearman coefficient, we determined a positive association between the two proteins (B). We also identified positive associations between integrin α5 and LTBP1 and NRP-1 (B). Further analyses of TCGA data sets with available miRNA expression data demonstrated an inverse association between miR-92 expression and integrin expression for alpha-5 and alpha-V subunits (C). A simple linear regression model was used to predict integrin expression as a function of miR-92a expression and provided 95% confidence intervals for our regression line. Spearman and Pearson correlation were performed using Stata 14 and coefficients have been provided (C, right).