Genomic loss of tumor suppressor miRNA-204 promotes cancer cell migration and invasion by activating AKT/mTOR/Rac1 signaling and actin reorganization

Abstract

Increasing evidence suggests that chromosomal regions containing microRNAs are functionally important in cancers. Here, we show that genomic loci encoding miR-204 are frequently lost in multiple cancers, including ovarian cancers, pediatric renal tumors, and breast cancers. MiR-204 shows drastically reduced expression in several cancers and acts as a potent tumor suppressor, inhibiting tumor metastasis in vivo when systemically delivered. We demonstrated that miR-204 exerts its function by targeting genes involved in tumorigenesis including brain-derived neurotrophic factor (BDNF), a neurotrophin family member which is known to promote tumor angiogenesis and invasiveness. Analysis of primary tumors shows that increased expression of BDNF or its receptor tropomyosin-related kinase B (TrkB) parallel a markedly reduced expression of miR-204. Our results reveal that loss of miR-204 results in BDNF overexpression and subsequent activation of the small GTPase Rac1 and actin reorganization through the AKT/mTOR signaling pathway leading to cancer cell migration and invasion. These results suggest that microdeletion of genomic loci containing miR-204 is directly linked with the deregulation of key oncogenic pathways that provide crucial stimulus for tumor growth and metastasis. Our findings provide a strong rationale for manipulating miR-204 levels therapeutically to suppress tumor metastasis.

Figure 1. Genomic loss of miR-204 in cancers.

A, high resolution miRNA-CGH on selected pediatric renal tumors with (left) or without (right) miR-204 deletions. Deletion of genomic loci containing the miRNA is indicated by dotted perpendicular lines. Red and green dots indicate position and value of each probe reflecting copy number change, represented in triplicate on the CGH array. The grey trend line represents the average value of the triplicate probe for each tumor. B, graphs obtained from meta-analysis of high-resolution CGH of ovarian cancers (n = 354; obtained from TCGA) representing a subset of tumors with or without deletion. The deletion of genomic loci containing miR-204 is indicated by dotted perpendicular lines. C and D, allelic PCR of miR-204 genomic locus in pediatric renal tumors (C) and ovarian cancers (D). The y-axis shows log₂ transformed relative quantification values. Dotted lines show the loss of copy threshold. E–G, graphical representation of qRT-PCR analysis showing levels of miR-204 in pediatric renal tumors (n = 38; E), in advanced stage ovarian cancers (n = 11; F) and, in breast cancers (n = 10; G), when compared to normal matched control kidney (n = 38), normal ovarian tissues (n = 5) and normal matched breast tissues (n = 10).

Figure 2. MiR-204 inhibits tumor growth and metastasis.

A, miR-204 inhibits anchorage-independent growth. Nutrient consumption (left) and graph (right) depict the number of colonies formed in soft agar wells by HEK-293 cells stably overexpressing either scramble or miR-204, further transfected with miR-204 inhibitor. (*) P<0.05; (**) P<0.01. Results are the average of three independent experiments. B, miR-204 overexpression inhibits tumor growth. Photograph shows representative features of tumor growth in RAG2 ^−/−, γc^−/− SCID mice injected (in the kidney capsule) with HEK-293 cells stably overexpressing either scramble control or miR-204. Bar graph shows mean tumor volume for miR-204 (n = 9) and scramble (n = 9) transfectants. (*) P<0.001. C, histological analysis of sections from tumor xenografts overexpressing either scramble (control) or miR-204. Images shown in the right panel represent magnified view of boxed region indicated in the left panel. Tumor invasion in control transfectants is reflected by the invasion of tumor into renal tissue. D, basement membrane matrix invasion assay of MDA-MB-231 cells transfected with 75 nM scrambled sequence (control) or miR-204 mimic (miR-204) or miR-204 mimic transfected cells further transfected with miR-204 inhibitor (miR-204+inhibitor). Bar graph shows the average number of invaded cells counted microscopically in five different fields per filter. (***) P<0.001.

Figure 3. Systemic delivery of miR-204 suppresses tumor metastasis.

A, injection of miR-204 oligonucleotide into tail vein suppressed lung metastasis. Live bioluminescence images of mice injected with miR-204 (n = 6) or miR-204 mutant (neg. control; n = 6) oligonucleotides using the Xenogen In Vivo Imaging System (IVIS) (Xenogen). Images were taken after subcutaneously injecting 150 mg/kg D-luciferin substrate in PBS to anesthetized mice. B, tumor metastasis volume was assessed starting from day 10 until animals were sacrificed at day 60. Using ROI analysis, tumor light intensity was calculated in photon/s, which corresponds with the number of live cells in vivo. C, representative lung images showing GFP^+ve foci (red circle) in neg. control groups. D, representative lung sections showing metastatic foci in neg. control groups. E, no hepatotoxicity in miR-204 injected mice. Sections of liver from miR-204 injected mice show no signs of hepatotoxicity. The presence of multifocal periportal lymphocytes is not unusual and is a common finding in young animals.

Figure 4. MiR-204 regulates expression of BDNF in cancers.

A–C, increased BDNF expression correlates strongly with lower miR-204 expression in multiple cancers. Graphical representation of qRT-PCR analysis showing the inverse correlation between miR-204 and BDNF in pediatric renal tumors (n = 38; A), advanced stage ovarian cancers (n = 11; B) and breast cancers (n = 10; C), compared to normal matched control kidney (n = 38), normal ovarian tissues (n = 5) and normal matched breast tissues (n = 10). D–H, BDNF is a bonafide target of miR-204. D, schematic of the putative miR-204 binding sequence in the BDNF 3′ UTR. E, HEK-293 cells were co-transfected with Renilla luciferase expression construct pRL-TK and firefly luciferase constructs containing either pMIR-BDNF 3′ UTR in the absence and presence of miR-204 mimic or pMIR-BDNF 3′ UTR mutant. Firefly luciferase activity of each sample was normalized to Renilla luciferase activity. Mean±SEM of three independent experiments (performed in duplicate for each experiment). (**) P<0.01; (***) P<0.001. F, qRT-PCR analysis of miR-204 overexpressing cells and cells transfected with miR-204 inhibitors using BDNF-specific primers. G, western blot analysis of HEK-293 cells transfected with miR-204 mimic using anti-BDNF antibody (1∶1000). β-actin was used as a loading control. Gel photographs are representative of three independent experiments. H, graphical representation of band intensities quantified using the Total Labs TL100 1D gel analysis software (n = 3; Nonlinear). BDNF protein level for the control was set to 100.

Figure 5. MiR-204 inhibits tumor cell migration and invasion by altering AKT/mTOR/Rac1 signaling.

A, miR-204 suppresses activation of AKT and mTOR signaling. HEK-293 cells transfected with miR-204 were grown in serum-free conditions and subjected to western blot analysis using anti-phospho-Ser⁴⁷³-AKT (1∶1000), anti-total-AKT (1∶1000), anti-phospho-Ser^235/236-S6 (1∶1000), anti-total-S6 (1∶1000), anti-phospho-Thr^37/46-4E-BP1 (1∶1000) and anti-total-4E-BP1 (1∶1000). β-actin (1∶10,000) was used as a loading control. Gel photographs are representative of three independent experiments. B, HEK-293 cells transfected with constitutively active AKT T308D/S473D mutant (CA-AKT) in the absence and presence of miR-204 were grown in serum free condition and subjected to western blot analysis as descried in A. C, overexpression of miR-204 abolishes BDNF-induced membrane ruffling and Rac1 translocation. HEK-293 cells transfected with miR-204 or miR-185 were grown in serum-free conditions and treated with or without BDNF (100 ng/mL) for 10 min and stained with Rac1 antibody (red) and FITC-phalloidin (green). Arrows indicate membrane-ruffling regions. D, miR-204 increases the sensitivity of HEK-293 cells to apoptosis as determined by Annexin V/PI staining using the FITC-Annexin V Apoptosis Detection Kit. The percentage cell population shown is the mean±SEM of three independent experiments. E, western blot analysis of HEK-293 cells transfected with miR-204 using anti-caspase-3 (1∶500) antibody and anti-PARP (1∶1000) show increased cleavage of caspase-3 and PARP. β-actin (1∶10,000) was used as a loading control. Gel photograph is representative of three independent experiments.