The scaffold protein IQGAP1 is crucial for extravasation and metastasis

IQGAP1 is a scaffold protein involved in a range of cellular activities, including migration, invasion, adhesion and proliferation. It is also oncogenic in a variety of cancers, promoting primary tumor growth and invasiveness. However, the role of IQGAP1 in tumor progression and metastasis remains unclear. In this study, we use both knockdown and knockout of IQGAP1 to investigate its role in the metastatic cascade of both melanoma and breast cancer cells in vivo. We find that reduction of IQGAP1 expression decreases the formation of both spontaneous and experimental metastases, without limiting primary or metastatic tumor growth. Furthermore, IQGAP1 knockout significantly inhibits extravasation of tumor cells from circulation, possibly involving invadopodial function. By expressing mutant forms of IQGAP1 in a knockout context, we also determine that IQGAP1’s pro-metastatic functions are dependent on multiple domains and functions. These data demonstrate that IQGAP1 is crucial for metastasis in vivo through regulation of extravasation and suggest that it may represent a valid therapeutic target for inhibiting metastasis.


IQGAP1 knockdown reduces metastasis.
To test the importance of IQGAP1 in metastasis in vivo, we first created stable IQGAP1 knockdowns in MA2 melanoma cells using miR30-based shRNAs (sh1 and sh6), along with control cells expressing shRNA against firefly luciferase (shFF) (Fig. 1A,B). Consistent with previous reports 22 , IQGAP1 knockdown caused cells to adopt a more spread and rounded morphology ( Supplementary  Fig. S1), along with a concordant reorganization of the actin and tubulin cytoskeletons ( Supplementary Fig. S2). Furthermore, as previously reported 18 , IQGAP1 knockdown significantly reduced the degradation of gelatin by cells in an in vitro assay for invadopodial activity (Fig. 1C).
To test whether IQGAP1 knockdown has any effect on metastatic ability in vivo, 50/50 mixes of red fluorescent control cells with green fluorescent cells expressing either control or IQGAP1-targeting shRNA were then injected into the tail veins of immunocompromised mice to observe differences in lung metastasis (Fig. 1D). Individual metastases from IQGAP1 knockdown cells were not significantly different in size compared to control metastases (Fig. 1E), consistent with the fact that IQGAP1 knockdown had no effect on proliferation in vitro ( Supplementary Fig. S3). However, IQGAP1 knockdown did reduce overall incidence of metastasis formation (Fig. 1F). Collectively, these results suggest that IQGAP1 expression is important in invasion and metastasis of tumor cells from circulation.

IQGAP1 knockout reduces metastasis but not primary tumor growth.
To create a clean genetic background, we next generated complete IQGAP1 knockouts in MA2 cells using CRISPR-Cas9. Four clonal lines were established using two different sgRNAs against IQGAP1, and stable expression of wild-type IQGAP1 was then re-established in each knockout line as a rescue control ( Fig. 2A), with rescue levels of IQGAP1 appearing somewhat higher compared to endogenous levels.
Each IQGAP1 knockout and rescue line was then assayed in vivo by tail-vein injection. Collectively, IQGAP1 knockout significantly reduced metastasis, while re-expressing wild-type IQGAP1 in the knockout lines restored metastatic ability (Fig. 2B), with individual results varying among the clonal knockout lines (Fig. 2C,D), consistent with the known variability observed to result from single-cell cloning in other cell lines 23 . These data broadly indicate that IQGAP1 knockout, like IQGAP1 knockdown, reduces metastasis of MA2 melanoma cells from circulation, whereas its overexpression increases metastasis.
To examine the relevance of IQGAP1 in metastasis in the context of a different cancer type, we also produced two clonal knockouts in LM2 breast cancer cells (an in-vivo-selected, highly metastatic derivative of MDA-MB-231) 24 , using the same two sgRNAs as above (Fig. 3A). These cells were then injected orthotopically into the mammary fat pads of immunocompromised mice to allow formation of primary tumors and ensuing metastasis. IQGAP1 knockout was total and was observed throughout the primary tumors in vivo, in contrast with wild-type tumors, which had extensive IQGAP1 expression ( Fig. 3B and Supplementary Fig. S4).
However, despite the complete lack of IQGAP1, there was no reduction in primary tumor growth; in fact, one of the clonal knockout lines had significantly increased primary tumor mass (Fig. 3C). We compared metastatic burden in the lungs and liver between the wild-type and IQGAP1 knockout cell lines (Fig. 3D) and observed an overall reduction in metastasis to both organs following the loss of IQGAP1 (Fig. 3E,F), implying that IQGAP1 has an effect on metastasis independent of primary tumor growth. IQGAP1 knockout thus reduced metastasis in vivo in both melanoma and breast cancer cell lines, indicating a more generally conserved role for IQGAP1 in the metastatic cascade. IQGAP1 knockout reduces extravasation. Metastasis from circulation, despite comprising only the latter half of the metastatic cascade, still requires a number of distinct steps to be completed before circulating tumor cells can grow into overt metastases, including arrest, extravasation, early survival and eventual outgrowth 25 . Of these steps, the importance of IQGAP1 in invadopodial activity (Fig. 1C) is of particular relevance to extravasation, since extravasation of cells from circulation has been shown to require invadopodia 26 . We therefore hypothesized that IQGAP1 may be exerting its pro-metastatic effects during extravasation.
To evaluate more directly at which stage of the metastatic cascade IQGAP1 expression may be crucial, we performed an in vivo extravasation assay by injecting MA2 cells into mouse tail veins, then collected lungs after 20 hours. By comparing ZsGreen cell staining and CD31 vascular staining using confocal microscopy, we quantified whether each cell remained entirely within the vasculature (non-extravasated) or had emerged (extravasated), and we calculated what fraction of tumor cells had extravasated by this time (Fig. 4A). For this and future experiments, we selected for further study one clonal knockout line (sg2a) whose knockout and rescued cellular phenotypes most closely matched those of IQGAP1 knockdown and wild-type cells, respectively. Compared with www.nature.com/scientificreports www.nature.com/scientificreports/ wild-type cells, the extravasation rate of IQGAP1 knockout cells was halved, while re-expression of wild-type IQGAP1 in the same knockout background completely restored extravasation ability (Fig. 4B). These results suggest that IQGAP1 expression is crucial for extravasation of MA2 cells, and that IQGAP1's effects on extravasation may account for much of its pro-metastatic ability in this system. IQGAP1's effects on metastasis require multiple domains. Finally, we sought to explore which domains and functions of IQGAP1 may be necessary for its effects on metastasis. In addition to its eponymous IQ motifs and RasGAP-related domain (GRD), IQGAP1 contains calponin homology (CH), coiled-coil (CC), WW and RasGAP C-terminal (RGCT) domains ( Fig. 5A) 27 . Of particular note, the IQ domain of IQGAP1 binds to the MAPK proteins B/C-Raf and Mek1/2 27 , as well as the metastasis promoter YAP 28 ; the GRD binds to the Rho GTPases Rac1 and Cdc42 27 ; and the RGCT binds to the exocyst complex proteins Sec3/8 18 . Any of these domains and binding interactions could plausibly be required for IQGAP1 to promote metastasis.
Consequently, we made MA2 cell lines expressing various mutant forms of IQGAP1 in the background of one of our clonal knockouts (sg2a): one mutant lacking all four IQ motifs (IQGAP1ΔIQ), another unable to bind Rac1 and Cdc42 (IQGAP1-T) 29 , a mutant lacking two major phosphorylation sites (IQGAP1-AA) and a final mutant missing the entire RasGAP C-terminal domain (IQGAPΔCT) ( Fig. 5A and Supplementary Fig. S5). When we observed these cells in culture, we noted that IQGAP1-T and IQGAP1-AA were able to revert knockout cells from a more spread morphology towards the elongated, wild-type MA2 cell shape, while IQGAP1ΔIQ and IQGAPΔCT had little effect on apparent cell morphology ( Supplementary Fig. S6).
Cell lines expressing these IQGAP1 mutants were then injected into mouse tail veins to assay for differences in lung metastasis (Fig. 5B,C). Consistent with our earlier results, we observed that IQGAP1 knockout reduced metastasis compared to wild-type cells, while re-expression of wild-type IQGAP1 rescued metastasis formation. However, expression of most of the IQGAP1 mutants (IQGAP1ΔIQ, IQGAP1-T and IQGAPΔCT) was unable to rescue metastatic ability. Only cells expressing IQGAP1-AA had metastasis formation restored to the level of wild-type cells. These results indicate that multiple domains of IQGAP1 are required for metastasis.

Discussion
IQGAP1 is involved in a myriad of pathways commonly associated with cancer and metastasis, including proliferation, migration and invasion. However, despite this accumulation of circumstantial evidence, direct proof of IQGAP1's importance in metastasis in vivo has been lacking. Furthermore, the diversity of roles performed by IQGAP1 could conceivably implicate it in any or all of the steps of the metastatic cascade: invasion from the primary tumor, intravasation, survival in circulation, extravasation, and outgrowth of the metastatic tumor. Lastly, given its enormous number of binding partners (well over a hundred) 21  www.nature.com/scientificreports www.nature.com/scientificreports/ of them, either individually or in combination, would be necessary for mediating any pro-metastatic effects. These broad uncertainties represent a diverse array of challenges in understanding the behavior and potential significance of IQGAP1 in metastasis.
In this study, we sought to begin the process of unraveling the complicated web of IQGAP1's interrelated functions as they relate to metastasis through the use of direct, in vivo experiments. Reduction in IQGAP1 expression, through both knockdown and knockout, reduced experimental metastasis of MA2 melanoma cells from circulation by two-to three-fold on average (Figs. 1F and 2B), while re-expression of IQGAP1 was generally able to rescue this metastatic ability, though it is possible that increased levels of IQGAP1 compared to endogenous could have contributed to this rescue. Furthermore, IQGAP1 knockout reduced spontaneous metastasis of LM2 breast cancer cells from primary tumors to both the lungs and liver by a similar or even greater degree (Fig. 3E,F), even when normalizing for any changes in primary tumor mass (Fig. 3C). These results clearly show that IQGAP1 expression is important for metastasis in these systems, and the consistency of this effect in both melanoma and breast cancer models implies that IQGAP1 may be broadly relevant in metastasis across multiple cancers.
If IQGAP1 is, indeed, regulating metastasis, then at which stage or stages of the metastatic cascade is it having an effect? Given that IQGAP1 knockdown and knockout both decrease experimental metastasis from circulation, IQGAP1 must at least be relevant in the later stages of the metastatic cascade, including arrest, extravasation, early survival and proliferation into a full metastatic tumor. Individual IQGAP1 knockdown metastases had a similar average size compared to wild-type metastases (Fig. 1E), suggesting that IQGAP1 is not having a marked effect on metastatic proliferation, consistent with in vitro data ( Supplementary Fig. S3), or else we would expect that IQGAP1 knockdown metastases would be significantly smaller. Instead, there is simply a diminished number of metastases forming. However, given IQGAP1's observed effects on invadopodia activity, and the reported importance of invadopodia in extravasation 26 , we hypothesized that IQGAP1 might be involved in metastasis at the extravasation step. We observed a two-to three-fold decline in extravasation in vivo of IQGAP1 knockout www.nature.com/scientificreports www.nature.com/scientificreports/ cells compared to either wild-type or IQGAP1 rescue cells (Fig. 4B). The magnitude of this change is comparable to that of the overall decrease in metastasis due to IQGAP1 knockout (Fig. 2B), suggesting that much, if not most, of the observed difference in metastasis is likely due to differential extravasation ability. These data do not rule out the possibility that IQGAP1 may also have a lesser but still substantial effect on arrest in circulation and early survival, particularly given its known regulation of cell-cell adhesion and MAPK signaling [14][15][16]30 . Furthermore, the fact that IQGAP1 knockout reduces spontaneous metastasis from primary tumors (Fig. 3E,F) at least as much, if not more, than it reduces metastasis from circulation (Fig. 2B) may mean that IQGAP1 has some additional influence in the early stages of the metastatic cascade. Given IQGAP1's effects on invadopodia (Fig. 1C) and extravasation (Fig. 4), this influence is most likely to apply during invasion of cancer cells from a primary tumor and subsequent intravasation into circulation. This is further supported by previous research demonstrating that IQGAP1 is overexpressed at the invasive front of human colorectal carcinomas 31 , and that breast cancer cells overexpressing IQGAP1 form more invasive tumors in mice 5 . Additional, detailed studies will be required to isolate more specifically all the individual steps of the metastatic cascade in which IQGAP1 expression is relevant. IQGAP1 may also promote tumor progression. In this study, we primarily reduced IQGAP1 expression in cell lines that were already highly proliferative and metastatic, and we did not see any reduction in primary tumor growth from LM2 breast cancer cells (Fig. 3C). However, previous results in the MCF-7 breast cancer cell line showed that altering IQGAP1 expression could affect primary tumor growth 5 . These results could be due to differences in the cell lines used: the LM2 line is much more aggressive than MCF-7, and knocking out IQGAP1 may not have a large-enough effect to cause an observable difference in growth. Additionally, the process of single-cell cloning that we used to generate our knockout cell lines may naturally select for cells that are more proliferative. IQGAP1 knockdown also shifted cell morphology from a more stretched towards a more spread and rounded appearance ( Supplementary Fig. S1), along with some cytoskeletal reorganization ( Supplementary  Fig. S2). These changes are consistent with morphological shifts observed in previous studies that demonstrated that changes in IQGAP1 expression can induce or reverse some forms of epithelial-mesenchymal transition (EMT) 8,22,32 . Moreover, IQGAP1 has been shown to bind to a number of known metastasis promoters, such as RhoC 33 and YAP 28 , which already have potent effects of their own. Thus, in addition to the marked role for IQGAP1 in extravasation that we have identified, this versatile scaffold protein may have further roles in directing tumor growth, regulating EMT and coordinating other known metastasis promoters.
Finally, given IQGAP1's enormous diversity of functions and binding partners, we attempted to identify which domains of IQGAP1 mediate its effects on metastasis by expressing several mutant forms of the protein in one of our clonal knockout MA2 lines (Fig. 5A, Supplementary Fig. S5). Only IQGAP1-AA, a mutant lacking two major phosphorylation sites 34 , was able to rescue metastasis to the level of wild-type cells, even slightly better than rescuing with wild-type IQGAP1 (Fig. 5B,C), indicating that phosphorylation at S1441 and S1443 is at least unnecessary for IQGAP1's effects on metastasis and may even act in opposition. Interestingly, IQGAP1-AA was shown to be deficient in promoting neurite outgrowth in neuroblastoma cells compared to wild-type IQGAP1 34 . This suggests that the function of these phosphorylation sites is particular to specific functions of IQGAP1, rather than globally controlling binding, as with the Ca 2+ -dependent association of Calmodulin with IQGAP1, which negatively regulates IQGAP1's other binding interactions 30,35,36 . Meanwhile, the remaining IQGAP1 mutants, those lacking all the IQ motifs (IQGAP1ΔIQ), those unable to bind Rac1 and Cdc42 (IQGAP1-T), or those missing www.nature.com/scientificreports www.nature.com/scientificreports/ the RasGAP C-terminal domain (IQGAPΔCT), were all unable to rescue metastasis in IQGAP1 knockout cells at all. The IQ motifs are responsible for binding the MAPK B/C-Raf 14 and Mek1/2 15 , as well as EGFR 37 and YAP 28 , involving this domain alone in cell survival, proliferation and migration. Indeed, the WW domain, which similarly binds the Erk1/2 MAPK, has previously been shown to be a valid therapeutic target: treatment with a peptide containing the IQGAP1 WW domain inhibited tumorigenesis and invasion of Ras-driven tumors in mice 6 . Moreover, YAP itself is a potent metastasis promoter 38 , so the interaction between IQGAP1 and YAP especially merits further investigation in the context of metastatic cancer. The inability of IQGAP1-T to regulate Rac1 and Cdc42 has very direct implications for cell migration and invasion, and cells expressing IQGAP1-T have been shown to be prone to form multiple leading edges 29 . It is therefore possible that the metastatic deficiency of cells expressing IQGAP1-T is due to their severely inhibited ability to extravasate out of blood vessels and invade into other tissues. However, the apparently low protein level of IQGAP1-T expressed in these cells ( Supplementary  Fig. S5) could mean that the inability of this mutant to rescue metastatic ability is due to insufficient expression, although the ability of even low levels of IQGAP1-T to alter cell morphology ( Supplementary Fig. S6) indicates that its expression is having some effect. The RGCT domain, much like the IQ domain, binds to a diverse array of proteins, including beta-catenin, E-cadherin, CLIP-170, APC and Sec3/8 39 . Thus, this domain is also critical in regulating proliferation, cell-cell adhesion, migration and invasion. This last function may be the most relevant in our study. The association between the exocyst complex proteins Sec3/8 and IQGAP1's RGCT domain is known to be crucial for the formation of mature invadopodia 18 , which are involved in both intravasation and extravasation 26,40 . Consequently, it is probable that the RGCT is directly involved in mediating IQGAP1's effects on extravasation (Fig. 4). Together, the failure of the individual IQGAP1ΔIQ, IQGAP1-T and IQGAPΔCT mutants to rescue metastasis to any significant degree implies that IQGAP1's effects in metastasis require several of its domains and functions, or at least a combination of binding partners that associate with different regions of the protein. More targeted studies, such as by blocking binding of specific proteins to IQGAP1, would be useful in isolating the particular binding partners important for metastasis.
In this study, we therefore present clear evidence that IQGAP1 is critical for metastasis in vivo, both in melanoma and breast cancer models. This need for IQGAP1 is due largely to effects on extravasation, though other steps of the metastatic cascade may also be influenced by IQGAP1. These effects on metastasis require multiple domains of IQGAP1, involving at least several of the numerous binding partners of this versatile scaffold protein.
The exact roles of IQGAP1 in metastasis merit further study, and IQGAP1 may represent a therapeutic target for metastatic cancer.

Methods
Cell lines. The human melanoma MA2 cell line 41  gift from David Benjamin; the original pHAGE vector backbone itself was a kind gift from Tyler Jacks). pHAGE-IQGAP1-IRES-Puro (wild-type IQGAP1) was cloned from IQGAP1 cDNA (GE Healthcare, accession BC139731). The pcDNA3-myc-IQGAP1-ΔIQ 42 and pcDNA3-myc-IQGAP1-S1441A/S1443A 34 vectors (referred to as IQGAP1ΔIQ and IQGAP1-AA in this study) were kind gifts from David Sacks. The pEGFP-IQGAP1-T1050AX2 (referred to in this study as IQGAP1-T, originally developed in the lab of Kozo Kaibuchi 29 ) and pEGFP-IQGAP1-T1050AX2-ΔCC + RGC 18 vectors were kind gifts from Philippe Chavrier. The T1050AX2 mutation was removed from IQGAP1-T1050AX2-ΔCC + RGC by cloning in the same region from wild-type IQGAP1, and the resulting construct is referred to as IQGAP1ΔCT in this study. MA2 cells were made green or red fluorescent through expression of pHAGE-ZsGreen-IRES-Hygro or pHAGE-TdTomato-IRES-Hygro, respectively (kind gifts from David Benjamin). Vectors for IQGAP1 knockdown and knockout are described in the section below. Retroviral and lentiviral production and transduction of cells was performed as previously described 43 .
Gene knockdown and knockout. miR30-based shRNAs targeting IQGAP1 for knockdown were designed using a tool developed by the lab of Michael Hemann (shrna.mit.edu) and cloned into MSCV-Puro-miR30, as previously described 38,43 . An shRNA against Firefly luciferase (shFF) was used as a control. sgRNAs for IQGAP1 knockout were designed using a tool developed by the lab of Michael Boutros (e-crisp.org) and cloned into lentiC-RISPRv2 44,45 . MA2 and LM2 cells were transiently transfected with lentiCRISPRv2 containing one of two sgRNAs against IQGAP1 (sg1 or sg2), then selected with puromycin. Cell populations were then sub-cloned to single cells.
Following expansion, clonal cell lines were tested for IQGAP1 expression by western blotting (see next section).
In this study, two clonal MA2 lines (a and b) were generated with each sgRNA (sg1 and sg2), resulting in four different clonal knockout lines (sg1a, sg1b, sg2a and sg2b). Each sgRNA was also used to generate a clonal LM2 line, resulting in two clonal knockout lines (sg1 and sg2). qRT-PCR analyses of all three IQGAPs in the knockout (MA2 and LM2) and rescue (LM2) lines showed insignificant levels of mRNA for IQGAP2 in any of these lines, in concordance with data from the human protein atlas (https://www.proteinatlas.org/ENSG00000145703-IQGAP2/ tissue) showing very low levels in skin and breast. Positive controls with RNA extract from human liver tissue confirmed the primers used. There were no significant changes in mRNA levels for IQGAP3, and IQGAP1 mRNA levels were as expected according to knockdown, knockout and rescue status (data not shown). Thus, these cell lines do not appear to be compensating for a lack of IQGAP1 by significantly upregulating either IQGAP2 or IQGAP3.

Immunoblotting, immunohistochemistry and quantitative PCR. For immunoblotting, cells were
Immunofluorescence and gelatin degradation assay. Immunofluorescence staining was performed as described elsewhere 47 , except that normal goat serum was used to block for goat secondary antibodies. TRITC-phalloidin (1:1000, Sigma) was used to detect actin, along with rabbit anti-α-tubulin (1:500, ab18251, Abcam). To label cover slips with fluorescent gelatin, 18 mm circular No. 2 cover glass (VWR) was washed with a 2:1 mixture of nitric to hydrochloric acid for 2 hr, rinsed with 70% ethanol, then coated with 50 µg/ml poly-D-lysine for 20 min and fixed with 0.5% glutaraldehyde for 15 min. After washing with PBS, cover slips were then coated with FITC-gelatin (Molecular Probes) mixed with 2% sucrose. Cover slips were then coated with 20 µg/ml fibronectin (Advanced BioMatrix) and quenched with 5 mg/ml sodium borohydride (Sigma). 30,000 cells were added to each gelatin-coated cover slip. Cells were fixed 5 hours after plating with 4% paraformaldehyde and stained with 0.5 µg/ ml DAPI (ThermoFisher). All images were taken with a Zeiss Axiovert 200 microscope using a 63× objective. Quantification of gelatin degradation area was conducted with ImageJ, and at least 50 cells were counted per line.
Proliferation assay. Cell proliferation was measured with the Incucyte Zoom System (Essen Bioscience).
10,000 cells were seeded in 10 replicate wells of a 96-well plate per cell line. Phase-contrast images were captured every 3 hours over 160 hours total to calculate percent confluence. 4 images were quantified per well per time point.