Studies using experimental tumor models have established a strong link between high levels of EMT activators and loss of cell polarity, reduced expression of basement membrane components, and increased propensity for metastasis (43
). The discovery that EMT activators endow epithelial tumor cells with pluripotency led to the current belief that metastatic propensity is directly related to plasticity in response to extracellular cues (12
). Here, positing that the scope of prometastatic biological processes controlled by ZEB1 extends beyond EMT and stem-ness, we discovered that ZEB1 drove promigratory cytoskeletal processes and metastasis by downregulating the expression of miR-34a. Exogenous miR-34a decreased tumor cell invasion and metastasis, inhibited the formation of promigratory cytoskeletal structures, suppressed activation of the RHO GTPase family, and regulated a gene expression signature that was enriched in cytoskeletal functions and prognostic in human lung adenocarcinomas. Biological reprogramming of this magnitude supports a central role for miR-34a in metastasis regulation by ZEB1.
RHO family members play key parts in the regulation of actin cytoskeletal remodeling and tumorigenesis. RAC1 is required for the development of primary lung adenocarcinomas in mice that express mutant KRAS (51
). The activities of RHO, RAC1, and CDC42 are coordinated to regulate membrane protrusions and cell-matrix adhesions at the leading edge of migrating cells to control forward movement (52
). Effectors of RHO GTPases include RHO-associated protein kinase, focal adhesions, and membrane protrusions, which together mediate cell adhesion to extracellular matrix, link matrix attachments to intracellular signaling pathways, and drive actomyosin contractility and cell locomotion (52
). Beyond these roles, a large body of evidence implicates RAC1 in the assembly, disassembly, and maintenance of adherens junctions and tight junctions, which play a central role in the regulation of apical-basal polarity (52
). Tightly regulating these processes is a miR network that targets RHO GTPases and their associated GEFs and GAPs (53
). Examples include RHOA
(miR-31, miR-133, and miR-155), RHOC
(miR-138 and miR-10b), CDC42
(miR-10b), and ARHGDIA
). Here we showed that miR-34a inhibited cytokine-induced RHO family GTPase activation and discovered that a RHOGAP, Arhgap1
, was a miR-34a target gene. ARHGAP1 reconstitution in miR-34a–overexpressing cells did not rescue RHO activation in response to EGF or TGF-β treatment, which was expected, given that RHOGAPs inhibit RHO GTPase activity. However, TGF-β–induced invasion was abrogated in metastasis-prone tumor cells by ARHGAP1 depletion and was rescued in miR-34a–overexpressing cells by ARHGAP1 reconstitution. The proinvasive effect of ARHGAP1 was paradoxical, given that RHO GTPase activity stimulates the formation of actin cytoskeletal structures that drive cell migration. Although the mechanism is unclear, ARHGAP1 binds to a number of proteins other than RHO family GTPases — including BNIP2, SRC, UBC, and PIK3R1 (BioGRID;
) — that regulate diverse biological processes and may have contributed to the proinvasive effect of ARHGAP1 through RHO GTPase–independent mechanisms. Collectively, these findings suggest that ARHGAP1 mediates some, but not all, of the biological effects of miR-34a (Figure H).
We discovered that ZEB1 regulated a larger number of miRs than had previously been reported (12
). This multiplicity was due in part to 19 miRs clustered within 7 genomic loci that are transcribed and processed together. ZEB1 downregulated certain miRs and upregulated others, which could be related either to the capacity of ZEB1 to function as a transcriptional repressor or activator (54
) or to indirect regulation of miRs by ZEB1. In support of the latter possibility, we found that ZEB1 indirectly repressed miR-34a through ΔNp63. The reported biological functions of the 46 miRs were diverse, encompassing hypoxic response (miR-210), cell differentiation (miR-326), proliferation (e.g., miR-224, miR-206, miR-542-3p, and miR-126), apoptosis (miR-96, miR-193a, and miR-181a), and migration (miR-206, miR-503, and miR-181b), among other functions (Supplemental Table 1), which indicates that ZEB1 might control a number of biological processes by regulating the expression of these miRs.
The p63 transcription factor family plays a central role in the regulation of embryonic development, normal adult tissue homeostasis, and malignancy (57
). The tumor-suppressive properties of TAp63 are exerted through the upregulation of a wide variety of miRs, including let-7, miR-15/16a, miR-145, miR-129, miR-26, miR-30, and miR-146a (57
). Senescence in keratinocytes is activated through ΔNp63-induced downregulation of miR-138, miR-181a, miR-181b, and miR-130b (58
). TAp63 is also a transcriptional activator of Dicer
, an endoribonuclease required for miR biogenesis (29
). The findings presented here build on this growing body of evidence that miRs are central mediators of the diverse biological actions of p63 by showing that miR-34a was upregulated by ΔNp63 and was a potent tumor suppressor in a Kras
-driven lung adenocarcinoma model. Furthermore, our finding that ΔNp63 served as a downstream mediator of ZEB1 completes a feedback circuit initiated by p63, which transcriptionally activates the miR-200b/a/429 cluster (59
) and, in turn, directly targets ZEB1 (9
), thereby relieving the ZEB1-induced repression of ΔNp63 shown here. There are numerous other p63/miR circuits, including one involving miR-193-5p, which targets p63 and is directly repressed by p63 (60
). Thus, miR homeostasis is tightly regulated through multiple mechanisms involving p63 and ZEB1.
The evidence presented here that miR-34a is a potent repressor of tumor growth and metastasis in a mouse model of human lung cancer bolsters evidence from other mouse models that miR-34a is a promising therapeutic agent. Delivery of miR-34a oligomers systemically by tail vein inhibits tumor growth in mice bearing lung adenocarcinomas, suppresses metastasis to the lung and other organs, and prolongs the survival of mice bearing orthotopic human prostate carcinomas (61
). The mechanisms by which miR-34a exerts its therapeutic effects are tumor cell type specific. For example, in the lung adenocarcinoma metastasis model shown here, miR-34a downregulation enhanced promigratory cytoskeletal processes, but was not required for stem cell features, based on formation of polarized epithelial spheres, whereas it targets the stem cell marker CD44
in prostate cancer cells and represses stem-ness in prostate, glioblastoma, pancreatic, and gastric cancer cells (18
). The distinct mechanisms by which miRs exert tumor suppressor functions in a given tumor type might be leveraged to create combinatorial treatment approaches. In metastatic KP cells, the miR-200 family members and miR-34a are all sharply downregulated, and ectopic expression of the miR-200b/a/429 cluster locks KP cells into an epithelial state and abrogates metastasis (17
). Thus, combined delivery of miR-34a and miR-200 family members might be complementary in these cells. Safe and efficient approaches using lipid-based nanoparticles (neutral or charged) have been developed that deliver miRs locally or systemically to the tumor tissue where they regulate their target genes (62
). Physical and chemical moieties of the particles that facilitate the targeted distribution and the controlled and sustained release of miRs are under clinical investigation (64
). External moieties, such as aptamers and ligands that enhance miR uptake by cancer cells, are being developed to direct the particles to a particular tissue (65
). Moreover, efforts are underway to initiate clinical trials that deliver miRs into patients with advanced cancer.