MicroRNA-7, a Homeobox D10 Target, Inhibits the p21-Aactivated Kinase 1 and Regulates its Functions

Cell lines, culture conditions and Transfections

Human cell lines were cultured as described in supplementary methods. Transfections for miRNA mimics and plasmids constructs were done with Oligofectamine or Fugene as described in the Supplementary methods.

Plasmid constructs, Luciferase assay and Western blotting

Pak1 3′- UTR and microRNA-7 promoter region were cloned into pGL3 control vector and pGL3 basic promote less vector respectively as described in supplementary methods. Luciferase assays and western blotting were done as described in the Supplementary methods.

Quantitative real-time PCR analysis of microRNAs

RNA was isolated using mirVana miRNA isolation kit. For quantitative analysis of miRNAs, two-step TaqMan real-time PCR analysis was performed using primers and probes obtained from Applied Biosystems.

Migration, Soft-agar and Confocal Studies

Migration, Invasion, Soft agar assays and Confocal studies were done as described in the Supplementary methods

Chromatin immunoprecipitation (ChIP)

Chromatin immunoprecipitation (ChIP) was performed as described in the Supplementary methods.

miR-7 targets 3′-UTR of Pak1 and inhibits its expression

In order to search for microRNAs that might regulate the Pak1 expression, we screened the 3′ -UTR region of human Pak1 mRNA against the public database for possible complementation of a minimum of 8-bp to the seed region of miRNAs (15). This exercise resulted in identification of miR-7 and miR-465 as candidate micro-RNAs for Pak1. MiR-7 is human miRNA (Fig. 1A) while miR-465 is of murine origin and both were predicted to target Pak1 in multiple species cells (Supplementary Fig. 1A). To directly evaluate the effects of miR-7 and -465 on Pak1 expressions, a number of human cancer cell lines were transfected with miR-7 or miR-465 or negative control (designated miR-con) miRNA mimics and expression of Pak1 levels were determined by the Western blotting. MiR-7 and miR-465 inhibited the levels of Pak1 protein and not Pak2, actin or vinculin (Fig. 1B and Supplementary Fig. 1B). To show the direct targeting of the 3′-UTR region by the miRs, we next cloned the 3′-UTR region of Pak1, complementary to miR-7 or miR-465, into the pGL3-luciferase reporter (Pak1 3′UTR-luc). We found that transfection of miR-7 or miR-465 along with respective Pak1 3′-UTR-luc into various human cancer cell lines resulted in a significant inhibition of luc-activity from the reporter, while there was no such inhibitory effect of the control miR-con (Fig. 1C and Supplementary Fig. 1C). These findings suggested that miR-7 or miR-465 directly targets 3′UTR of Pak1 and selectively inhibits the Pak1 expression. To assess the relationship between the endogenous levels of Pak1 and its miRs, we next determined the levels of miR-7 and miR-465 by quantitative polymerase chain-reaction (q-PCR) as well as Pak1 protein in a variety of exponentially growing cancer cell lines. In general, cells with increased levels of miR-7 contain reduced levels of Pak1 protein as compared to cells with low levels of miR-7 and high levels of Pak1 (Fig. 1D). Inspite of repeated attempts, we were unable to detect miR-465 in human cells (data not shown), presumably due to its restricted expression in murine systems. Therefore, we have studied the regulation of Pak1 expression and functions by miR-7 in the subsequent studies.

MiR-7 is directly regulated by the transcription factor HoxD10

To gain insights into the mechanisms by which miR-7 might be regulated in physiologic setting, we analyzed the upstream 2 kb-region of the putative miR-7 promoter for the presence of binding motifs for various transcription factors using the PROMO (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3 and http://www.fruitfly.org/seq_tools/promoter.html) search programs. This analysis suggested that miR-7 resides within the first intron of heterogenous ribonuclear protein K gene on chromosome 9. We found the presence of two perfect consensus motifs for HoxD10 within the -958 to -968 and -1019 to -1028 from the transcriptional initiation site of miR-7 (Fig. 2A). Since HoxD10 has been intimately linked with the invasion and metastatic potential of human breast cancer cells (6, 18) and because HoxD10 expression is lost as a function of invasion (18) and reintroduction of HoxD10 restores non-tumorigenic phenotypes in invasive breast cancer cells (18), we decided to focus on HoxD10 as a potential upstream modular of miR-7 expression.

To study the impact of HoxD10 on the expression of miR-7, we next cloned the putative miR-7 promoter into a TATA-less basic pGL3-luc reporter (pGLmiR-7) and established the functionality of pGLmiR-7 in the HeLa cells (Fig. 2B). We next demonstrated that transient expression of HoxD10 efficiently but not control vector stimulates the transcription of miR-7 from the pGLmiR-7 reporter in multiple human cancer cell lines (Fig. 2C). To demonstrate the potential recruitment of HoxD10 to the miR-7 gene, we next performed chromatin immunoprecipitation analysis (ChIP) of the miR-7 chromatin. Due to the lack of a good ChIP-grade HoxD10 antibody, we used a myc-tagged HoxD10 expression vector in these studies. We found that Myc-HoxD10 is indeed, recruited to miR-7 promoter region encompassing from -1139 to −899 (240 bp) which included both HoxD10 binding sites. PFK promoter was used as a negative control, which lacks HoxD10 binding sites (Fig. 2D). Together, these observations suggested that HoxD10 directly interacts with the putative miR-7 promoter and that miR-7 expression is positively regulated by HoxD10 transcription factor.

HoxD10 regulation of Pak1 expression via miR-7

Since miR-7 targets Pak1 (this study) and because HoxD10 positively regulates the expression of miR-7 (this study) and the fact that the loss of HoxD10 (6, 18) is a major contributor of cancer cell invasiveness which is also driven by Pak1 (10, 19), the above findings suggested an interesting possibility of a dynamic relationship between the Pak1, miR-7 and HoxD10, and potential role of HoxD10 in the cellular action of the miR7-Pak1 axis in cancer cells. To examine these possibilities in the context of breast cancer progression to more aggressive phenotypes, we next analyzed the levels of Pak1, miR-7, and HoxD10 in exponentially growing cell lines derived from the MCF-10A model system (20). This model consists of four isogenic, human breast cancer cell lines with distinct invasive and metastatic potential. For example, MCF-10A cells are non-malignant human breast cancer cells; MCF10AT cells are weakly tumorigenic; MCF-10CA cells are undifferentiated metastatic carcinomas; and MCF10DCIS cells are highly proliferative, aggressive and invasive in nature (20, 19). Interestingly, we found that the levels of Pak1 protein progressively upregulated while that of miR-7 as well as HoxD10 are progressively downregulated from low invasive MCF10A to highly invasive MCF-10DCIS cells (Fig. 3A). To validate these results, we next showed that transient over expression of HoxD10 leads to upregulation of miR-7 (Fig. 3B) and downregulation of the Pak1 3′UTR-luc activity in the highly invasive MCF-10DCIS cells as well as in the HEK-293 cells (Fig. 3C). The noticed regulation of miR7 and Pak1-UTR activity by HoxD10 was also accompanied by a corresponding down regulation of Pak1 protein in HEK-293 and MCF10DCIS cells (Fig. 3D). In brief, these results suggested a regulatory role of HoxD10 in modifying the expression of miR-7, and consequently, functions of miR-7-Pak1 pathway in human cancer cells.

MiR-7 suppresses motility, invasiveness and anchorage-independence of breast cancer cells

To further delineate the potential effects of miR-7 downregulation of Pak1 on the biology of breast cancer cells, we next examined the effects of miR-7 on the biology of MDA-MB231 cells, an established model system for highly invasive and tumorigenic breast cancer cells. We found that miR-7-mediated downregulation of Pak1 in MDA-MB231 cells were accompanied by a profound inhibition of the cell motility, the cell invasiveness, the ability of cells to grow in an anchorage-independent manner and inhibited tumorigenic potential in nude mice (Fig. 4A-C). These findings raise the possibility that miR-7 could be potentially developed as an agent to downregulate the expression, and consequently, tumor-promoting functions of Pak1. This is important as Pak1 or its effectors have been shown to be upregulated in a number of human cancers, including breast and gliomas (10-12). Accordingly, a number of academic groups and pharmaceutics have initiated research programs to target Pak1 kinase activity for cancer therapy (13, 14).

While this study was near completion, we noticed an interesting report describing the ability of miR-7 to also inhibit the EGFR and IRS1 by directly targeting the 3′-UTR regions of its mRNAs (17). This is understandable but exciting, given the fact that miRs are expected to target multiple mRNAs with desired complementary base-pair matching in their 3′-UTR, and thus, reaffirming the emerging regulatory role of miR-7 in cancer cells. Furthermore, microRNAs are expected to work as part of network and could affect the components of the same pathway at multiple levels (7). Since Pak1 signaling is also affected EGFR (10) and Akt (a target of IRS1), we next examined the levels of EGFR and IRS-1 proteins in the cell lysates used above in this study. As expected, transfection of miR-7 downregulates the levels of EGFR and IRS1 proteins in the Hela, ZR-75 and MDA-231 cells (Supplementary Fig. 2). We further observed that the levels of EGFR and IRS1 correlate well with the levels of Pak1 but inversely related with the endogenous miR-7 status in human cell lines with the exception of IRS1 in the MCF-10A model system (Supplementary Fig. 3A, B). Interestingly, we found that HoxD10 overexpression in the HEK-293 or MCF-10DCIS cells also leads to a distinct downregulation of EGFR and IRS-1 (Supplementary Fig. 4), presumably due to increased expression of miR-7 (this study, Fig. 3B).

In summary, the findings presented here in conjunction of recent reports on miR-7 and HoxD10 suggest a model wherein HoxD10, a known regulator of cancer invasiveness (6, 18), is likely to control the levels of Pak1 protein (and of EGFR and IRS1 and perhaps, other targets yet to be described) and thus, impact the cancerous phenotypes, via miR-7 pathway. Since reintroduction of HoxD10 into MDA-MB231 cells have been previously shown to compromise invasiveness (18), these results now suggest a potential role of the noted reduction in the Pak1 level due to increased expression of miR-7. In addition, our present findings also connect the presently described HoxD10/miR-7/Pak1 pathway with a recently described role of invasion promoting role of Twist regulation of miR-10B which in-turn, has been shown to inhibit the translation of HoxD10 (6). In the light of observations presented here, it is possible that inhibition of HoxD10 will lead to repression of miR-7 level and upregulation of Pak1 expression and its functions (Fig. 4D). In order to have a comprehensive understanding of the emerging significance of microRNA-network and engagement of multiple targets in shaping the behavior of the cancer cells, it will be important to keep investigating the above hypotheses and testing the lead microRNA in more complex model systems in due course.