IQGAP1 regulates TβRII abundance in HSCs.
Since TβRII is the most upstream receptor that initiates TGF-β signaling, we tested to determine whether IQGAP1 associated with TβRII and regulated TβRII in human primary HSCs (23
). To this end, we first validated the specificity of anti-TβRII antibody by Western blot (WB) analyses, since the quality of commercial anti-TβRII is variable (Supplemental Figure 1; supplemental material available online with this article; doi:
). Using this antibody, we found that IQGAP1 regulates TRII abundance in HSCs (Figure A). To avoid the possibility of off-target effects of shRNA, multiple IQGAP1 shRNAs (Sigma-Aldrich), each targeting a distinct sequence of human IQGAP1, were used to knock down IQGAP1. In cells expressing TβRII-HA, IQGAP1 knockdown increased TβRII protein levels and overexpression of IQGAP1 decreased TβRII (Figure A). Additionally, IQGAP1 shRNAs also increased endogenous TβRII protein levels (Figure A). Thus, Iqgap1
activity reduces levels of TβRII protein in HSCs.
IQGAP1 interacts with TβRII and regulates its stability.
IQGAP1 interacts with TβRII in HSCs.
Quantitative real-time RT-PCR revealed that IQGAP1 knockdown did not influence TβRII mRNA levels (Figure B), suggesting that IQGAP1 regulates TβRII stability at the posttranscriptional level, possibly by binding to TβRII and promoting its degradation. To test this hypothesis, we performed double immunofluorescence staining (IF) for IQGAP1 and TβRII and found that IQGAP1 and TβRII colocalized at the peripheral plasma membrane (arrowheads, Figure C) and in endocytic vesicles (arrows, Figure C) in cells expressing TβRII-HA. Coimmunoprecipitation (IP) also demonstrated that these 2 proteins coprecipitated in HSCs expressing TβRII-HA (Figure D). Furthermore, IQGAP coprecipitated with endogenous TβRII from cells as well (Figure D). These data suggest that IQGAP1 interacts with TβRII in HSCs. Additionally, the interactions between these 2 proteins occur in other cell types as well (Supplemental Figure 5).
IQGAP1 aa 1503–1657 is required for binding and suppressing TRII.
IQGAP1 contains multiple protein-protein interacting domains including calponin-homology domain (CHD), poly-proline protein-protein domain (WW), IQ domain (IQ), Ras GTPase-activating protein–related domain (GRD), and RasGAP C terminus (RGCt) (Figure A and ref. 9
). So we performed in vitro glutathione-S
-transferase (GST) pull-down assays to map the TβRII-binding region on IQGAP1. Both aa 746–1657 and aa 1503–1657 of IQGAP1 interacted with TβRII by GST pull-down assays (Figure A), suggesting that the TβRII-binding region is within the smaller C-terminal 1503–1657 fragment. To understand whether IQGAP1/TβRII binding is direct or requires adaptor proteins, we performed in vitro protein-binding assays by incubating detagged TβRII (the GST tag was removed by thrombin) with GST-fused IQGAP1 proteins (Figure B), or detagged IQGAP1 with GST-fused TβRII (Figure B and Supplemental Figure 2). Both experiments demonstrated a direct binding of these 2 proteins in vitro.
IQGAP1 C terminus aa 1503–1657 is required for binding and suppressing TβRII.
To test the role of aa 1503–1657 of IQGAP1 in IQGAP1/TβRII binding and TβRII abundance, we generated a IQGAP1 (1-1502) mutant that lacks aa 1503–1657 and found that this mutant failed to suppress TβRII protein levels in contrast with full-length IQGAP1 (Figure C). Thus, IQGAP1 aa 1503–1657 is required for IQGAP1/TβRII binding and suppressing TβRII. Interestingly, the C-terminal region of IQGAP has previously been shown to bind to β-catenin and other molecules contained within key signaling nodes (16
), suggesting a potentially important biological significance of IQGAP binding with TβRII.
IQGAP1 suppresses TGF-β1–mediated activation of pericytes into myofibroblasts.
Since receptor stability and trafficking importantly regulate signaling, we tested the significance of IQGAP1/TβRII binding on myofibroblastic activation of HSCs. Two different siRNAs (QIAGEN) were used to knock down IQGAP1 of HSCs. Cells were stimulated with TGF-β1 (5 ng/ml) or PDGF-BB (20 ng/ml) and myofibroblastic activation of HSCs was assessed by WB for α-SMA, fibronectin, and phospho-SMAD2 (p-SMAD2). TGF-β1 more prominently activated HSCs as compared with PDGF-BB, as determined by upregulation of α-SMA, fibronectin, and p-SMAD2 (Figure A). IQGAP1 knockdown by 2 distinct IQGAP1 siRNAs also consistently potentiated TGF-β1 activation of HSCs (Figure A).
IQGAP1 suppresses TGF-β–mediated activation of HSCs into myofibroblasts.
Double IF for IQGAP1 and α-SMA demonstrated that IQGAP1-knockdown cells exhibited prominent α-SMA–positive stress fibers, indicative of myofibroblastic transdifferentiation (arrows, Figure B). Quantitative data from cells stimulated with TGF-β1 revealed that IQGAP1 siRNA increased TGF-β1–induced myofibroblastic activation by 35% (Figure C). Moreover, a SMAD siRNA targeting both SMAD2 and SMAD3 abolished the effect of IQGAP1 siRNA on myofibroblastic activation (Supplemental Figure 3). As expected, overexpression of full-length IQGAP1 suppressed HSC activation and the IQGAP1 (1-1502) mutant failed to repress HSC activation (Figure D). Taken together, these data demonstrate that by binding to TβRII, IQGAP1 suppresses TGF-β1/SMAD–mediated myofibroblastic activation of HSCs in vitro.
TGF-β1 stimulation increases IQGAP1/TβRII binding.
TGF-β1 ligand induces internalization and downregulation of TβRII (24
). Therefore, we tested the hypothesis that IQGAP1 may modulate ligand-dependent internalization and degradation of TβRII in HSCs. To this end, we performed IP using anti-IQGAP1 and TβRII WB to detect TβRII/IQGAP1 binding. As shown in Figure A, TGF-β1 induced temporal increase of IQGAP1/TβRII binding, supporting a model whereby TGF-β1 stimulation recruits IQGAP1 to TβRII-containing signaling complexes, and in turn, IQGAP1 may modulate TβRII trafficking, degradation, and TGF-β1 signaling.
TGF-β1 increases IQGAP1/TβRII binding, and IQGAP1 knockdown inhibits lysosomal targeting of TβRII.
IQGAP1 knockdown inhibits lysosomal targeting of TβRII and induces accumulation of TβRII in the early endosomes.
TβRII was localized to endosomes and its degradation was attenuated by lysosomal inhibitors (27
), so we tested to determine whether IQGAP1 knockdown could alter the trafficking of TβRII to endosomes and lysosomes, 2 intracellular compartments where signaling and receptor turnover are regulated (31
). HSCs treated with TGF-β1 were subjected to double IF for HA-tagged TβRII and lysosomal-associated membrane protein 1 (LAMP1, late endosome/lysosomal marker) or early endosome antigen 1 (EEA-1, early endosomal marker). IQGAP1 IF confirmed IQGAP1 knockdown in HSCs (Supplemental Figure 4A). Double IF revealed that at both 30 and 60 minutes after TGF-β1 stimulation, IQGAP1 knockdown significantly reduced TβRII reaching LAMP1-positive vesicles (late endosome/lysosomes) (arrowheads, Figure , B and C). In control cells, TβRII/EEA-1 colocalization increased at 5 minutes after TGF-β1 stimulation and decreased gradually thereafter (Figure , D and E). In IQGAP1-knockdown cells, however, TβRII/EEA-1 colocalization continuously increased at 30 or 60 minutes after TGF-β1 stimulation (Figure , D and E), suggesting that IQGAP1 knockdown induces accumulation of TβRII in the early endosomes.
IQGAP1 knockdown inhibits lysosomal and proteasomal degradation of TβRII.
We next used biotinylation of cell-surface proteins to analyze TGF-β1 downregulation of cell-surface TβRII in control and IQGAP1-knockdown cells. In control cells, TGF-β1 downregulated cell-surface TβRII in a time-dependent manner; TβRII half-life was about 44 minutes (Figure A). In IQGAP1-knockdown cells, however, it increased to about 63 minutes (Figure A), consistent with the observation that IQGAP1 knockdown inhibited TGF-β1–mediated lysosomal targeting of TβRII. Additionally, both chloroquine (Chlo, lysosomal inhibitor) and MG132 (proteasomal inhibitor) were able to partially prevent TβRII downregulation (Figure A). Furthermore, IQGAP1 knockdown also inhibited TGF-β1 downregulation of total cellular TβRII protein in cells that were pretreated with cycloheximide (Supplemental Figure 4B). Thus, these data support a model that IQGAP1 binds to TβRII and promotes TGF-β1–mediated lysosomal and proteasomal degradation of TβRII.
IQGAP1 knockdown inhibits TGF-β1 downregulation of TβRII, TβRII ubiquitination, and the plasma membrane targeting of SMURF1.
IQGAP1 knockdown inhibits TβRII ubiquitination.
Since TGF-β stimulation induces the formation of complexes that contain TβRII and TβRI (5
), we compared TβRI protein levels in control and IQGAP1-knockdown HSCs. Similar to TβRII, IQGAP1 knockdown also increased exogenously expressed TβRI-FLAG in HSCs (Figure B), further supporting the model whereby IQGAP1 is recruited to the TGF-β receptor complexes where it promotes the degradation of TGF-β receptors.
Ubiquitination is an important signal for plasma membrane receptor internalization, multivesicular body sorting, and degradation (36
). TβRII is also subjected to ubiquitin modification similarly to TβRI (33
), so we tested to determine whether IQGAP1 knockdown influenced the ubiquitination of TβRII. To this end, TβRII-HA was precipitated from HSCs expressing TβRII-HA, and TβRII ubiquitination was detected by ubiquitin WB. As shown in Figure C, IQGAP1 knockdown markedly reduced the ubiquitination of TβRII in HSCs.
IQGAP1 is required for the targeting of SMURF1 to the plasma membrane.
The turnover of TGF-β receptors is regulated by the E3 ubiquitin ligases such as SMURF1 and SMURF2, which interact and ubiquitinate TGF-β receptors at the plasma membrane (27
). Based on this model, we tested to determine whether IQGAP1 knockdown influenced the subcellular localization of SMURF1. Consistent with the concept that SMURF1 localizes at the cellular protrusions (40
), we found that in control HSCs, SMURF1 localized at the peripheral plasma membrane in addition to the nucleus and cytoplasm (arrows, Figure D). IQGAP1 knockdown reduced SMURF1 at the plasma membrane (Figure D) and SMURF1/TβRII colocalization at the plasma membrane (Figure E). Interestingly, we also found that IQGAP1 knockdown reduced the total protein levels of SMURF1 (Figure F), suggesting a role of IQGAP1 in the regulation of SMURF1 stability. Thus, IQGAP1 promotes the ubiquitination and degradation of TβRII in HSCs possibly by at least 2 different mechanisms: (a) directing SMURF1 to the plasma membrane where SMURF1 interacts with the TGF-β receptor complexes and (b) stabilizing SMURF1 protein levels.
Evidence for a basal activation phenotype of HSCs of Iqgap1–/– mice.
To determine whether IQGAP1 suppresses HSC activation in vivo, we isolated livers from 1-year-old Iqgap1+/+
mice for IF and WB. As compared with matched Iqgap1+/+
livers, double IF revealed that Iqgap1–/–
livers contained significantly more HSCs that were double-positive for α-SMA and desmin, another marker of HSCs (refs. 41
, and Figure A). WB confirmed this morphologic observation (Figure B). Additionally, Iqgap1–/–
livers contained significantly more collagen I, as detected by WB (Figure B). Next, we isolated HSCs from mice and treated them with TGF-β1 for 24 hours and found that Iqgap1–/–
HSCs exhibited an enhanced activation phenotype in vitro as compared with Iqgap1+/+
HSCs (Figure C). Thus, these data support that IQGAP1 of HSCs suppresses HSC activation in vivo.
Basal activation phenotype of HSCs of Iqgap1–/– mice.
IQGAP1 deficiency in the tumor microenvironment promotes myofibroblastic activation and liver metastatic growth.
The basal activation phenotype of HSCs of Iqgap1–/–
mice led us to test if Iqgap1–/–
livers promoted liver metastatic growth. Lewis lung carcinoma cells (LLCs), a mouse cancer cell line that is widely used in metastasis studies, were implanted into the livers of Iqgap1+/+
mice by portal vein injection (Figure A). This study allowed us to study the specific effect of IQGAP1 depletion in the liver microenvironment on liver metastatic growth, since the implanted LLCs harbored intact IQGAP1 protein. Upon necropsy, we found that the average tumor weight in the liver of Iqgap1–/–
mice was 4 times greater than that of Iqgap1+/+
mice at 10 days after implantation (Figure B) (Iqgap1+/+
: 183.8 ± 72 mg/liver; Iqgap1–/–
: 832.7 ± 255 mg/liver; P
< 0.05), indicating that IQGAP1 deficiency in the tumor microenvironment promotes liver metastatic growth in mice. Since Iqgap1–/–
T cells do not exhibit reduced cytolytic function as compared with Iqgap1+/+
T cells (43
), this enhanced liver metastatic growth phenotype of Iqgap1–/–
mice is unlikely to be due to IQGAP1 depletion in T cells.
IQGAP1 deficiency in the liver promotes myofibroblastic activation and lung liver metastases in mice.
Liver metastases isolated from the livers were subjected to WB and IF for α-SMA (maker of tumor-associated myofibroblasts) and PECAM-1/CD31 (marker of endothelial cells). As revealed by WB, the average level of α-SMA protein in the liver metastases of Iqgap1–/– mice was more than 10 times higher than that of Iqgap1+/+ mice (P < 0.01) (Figure C). Consistent with our previously depicted in vitro data, the average level of TβRII protein in the liver metastases of Iqgap1–/– mice was more than 3 times higher than that of Iqgap1+/+ mice (P < 0.05) (Figure C). In contrast, PECAM-1/CD31 protein levels were comparable in liver metastases of both groups (Figure C). IF confirmed that the liver metastases of Iqgap1–/– mice indeed contained more α-SMA–positive tumor-associated myofibroblasts (Figure D) and that endothelial cell densities were comparable in both groups (Supplemental Figure 6). Taken together, this liver metastasis study demonstrates that IQGAP1 in mesenchymal cells residing in the tumor microenvironment suppresses TβRII protein levels, myofibroblastic activation in vivo, and liver metastatic growth.
IQGAP1 deficiency in the tumor microenvironment promotes colorectal liver metastases.
Since gastrointestinal cancers including colorectal and pancreatic cancers show a preference for liver metastasis, we next implanted MC38 mouse colorectal cancer cells into the livers of Iqgap1+/+ and Iqgap1–/– mice. Similar to LLCs, MC38 cells implanted, quickly multiplied, and occupied mouse liver in vivo. The median survival of Iqgap1+/+ mice was about 19 days, and it was shortened to 13 days for Iqgap1–/– mice (P < 0.01), confirming an enhanced colorectal liver metastatic growth phenotype in Iqgap1–/– mice (Figure A). Next, MC38 cells expressing firefly luciferase were implanted to determine whether IQGAP1 deficiency in the tumor microenvironment promoted tumor implantation into the liver. In vivo xenogen imaging that measured bioluminescence of MC38 cells revealed that at day 3 after tumor implantation, significantly more MC38 cells were detected in Iqgap1–/– livers than in Iqgap1+/+ livers (Figure , B and C). These data indicate that Iqgap1–/– livers promote colorectal tumor implantation possibly by protecting the tumor cells from anoikis. This hypothesis was pursued as shown below.
IQGAP1 deficiency in the liver promotes colorectal liver metastases in mice.
IQGAP1-knockdown HSCs promote colorectal tumor implantation and growth in mice.
In addition to HSCs, other liver-resident cells, such as fibroblasts, bone marrow–derived fibrocytes, hepatocytes, or cholangiocytes after epithelial-mesenchymal transition, are postulated to play a role in liver fibrosis (44
). Therefore, we next performed an HSC/tumor cell coimplantation study to define a specific and selective role of HSCs for myofibroblastic activation and tumor growth. HT-29 human colorectal cancer cells that were mixed with an equal number of control HSCs (transduced with NT shRNA) or IQGAP1-knockdown HSCs (transduced with IQGAP1 shRNA) were implanted into nude mice via subcutaneous injection. Tumor growth curves generated by monitoring mice carefully for 17 days revealed that both control and IQGAP1-knockdown HSCs accelerated HT-29 tumor growth in mice (Figure A). Furthermore, IQGAP1-knockdown HSCs exerted a greater effect on promoting HT-29 tumor growth as compared with control HSCs (Figure A). Since HT-29 cells were tagged by firefly luciferase before implantation, in vivo xenogen imaging was performed to study the role of IQGAP1-knockdown HSCs in HT-29 implantation in mice (Figure B). At day 5 after implantation, the HT-29/HSC-IQGAP1 shRNA coimplantation group exhibited the highest level of HT-29 bioluminescence as compared with other groups (Figure B). A detailed analysis revealed that in the HT-29–only implantation group, HT-29 bioluminescence decreased continuously at days 3 and 5 after implantation, possibly representing anoikis of HT-29 cells, and that coimplantation of either control or IQGAP1-knockdown HSCs increased HT-29 bioluminescence at these time points (Figure B). Furthermore, coimplantation of IQGAP1-knockdown HSCs resulted in the greatest increase of HT-29 bioluminescence (Figure B). Thus, this HSC/tumor cell coimplantation study supports the concept that IQGAP1-knockdown HSCs promote colorectal tumor growth by promoting the implantation of tumor cells in mice.
IQGAP1-knockdown HSCs promote colorectal tumor implantation and growth in HSC/tumor coimplantation model.
IQGAP1 knockdown in HSCs promotes TβRII protein levels and myofibroblastic activation of HSCs in mice.
To understand whether coimplanted HSCs indeed transdifferentiated into tumor-associated myofibroblasts in mice, we performed in vivo xenogen imaging to determine the survival of the coimplanted HSCs. HSCs tagged with firefly luciferase were implanted into nude mice alone (control) or with HT-29 cells via subcutaneous injection. In the HSC-only implantation group, bioluminescence of HSCs started to decrease continuously at day 6 to an undetectable level at day 14 after implantation (Figure C). In the HSC/tumor cell coimplantation group, however, it increased again at day 13 and remained at a detectable level at day 23 after implantation (Figure C; data are representative of 6 mice with consistent results). These data indicate that HSCs are able to survive up to 23 days after HSC/tumor cell coimplantation and that this prolonged survival of HSCs is dependent on tumor cells. Since the coimplanted HSCs were also tagged by TβRII-HA fusion proteins, we performed double IF on isolated tumor nodules to visualize the coimplanted HSCs. As revealed by double IF for HA tag and α-SMA, most HA-positive cells in the tumor nodules also expressed α-SMA (arrows, Figure A), suggesting that these coimplanted HSCs indeed transformed into tumor-associated myofibroblasts. WB revealed that the average level of TβRII-HA or α-SMA in tumors arising from IQGAP1-knockdown HSC coimplantation was significantly higher than that in tumors arising from control HSC coimplantation (Figure B). This finding was confirmed by IF for HA (Figure C) and α-SMA as well (Figure D). Thus, this HSC/tumor cell coimplantation study demonstrates a suppressive role of HSC IQGAP1 for TβRII protein, myofibroblastic activation, and tumor growth in vivo.
IQGAP1 knockdown in HSCs promotes TβRII levels, myofibroblastic activation of HSCs in a HSC/tumor coimplantation model.
HSCs are activated into tumor-associated myofibroblasts of liver metastases.
To determine whether HSCs in the liver indeed transdifferentiate into the tumor-associated myofibroblasts of liver metastases, we performed portal vein implantation of L3.6 human gastrointestinal cancer cells into the liver of SCID mice and isolated xenografts for IF. Stem121 is an antibody that has been extensively used to detect the engraftment of human cells transplanted into mice owing to its ability to detect a cytoplasmic protein specific to human origin cells. Double IF for α-SMA and Stem121 revealed that the stroma (S) of liver metastases identified by α-SMA–positive staining were negative for Stem121 and that tumor cells (T) were positive for Stem121 and negative for α-SMA (Figure A). These data indicate that these tumor-associated myofibroblasts were not derived from the implanted human cancer cells, but rather, from cells residing in the host mouse liver. To identify their origin, immunohistochemistry for α-SMA and desmin was perform on adjacent sections of the liver metastases. As shown in Figure B, some of these stromal cells were indeed positive for desmin, suggesting that they may have an HSC origin. To test this hypothesis further, we performed double IF for α-SMA and desmin on liver sections containing L3.6 micrometastases and found that a fraction of HSCs adjacent to the L3.6 tumor cells were positive for both α-SMA and desmin (arrowheads, Figure C). Additionally, these activated HSCs were negative for Stem121 (arrows, Figure D). It is interesting that L3.6 cells were also positive for desmin, with desmin representing one of a panel of diagnostic markers for certain tumors (45
). Taken together, these data provide evidence for transactivation of liver-resident HSCs into the tumor-associated myofibroblasts using an experimental liver metastasis model.
HSCs are activated into tumor-associated myofibroblasts of liver metastases.
IQGAP1-knockdown HSCs confer a greater stimulatory effect on proliferation, migration, and survival of tumor cells.
To understand mechanisms by which IQGAP1-knockdown HSCs promoted liver metastatic growth in mice, conditioned medium were collected from control and IQGAP1-knockdown HSCs and incubated with tumor cells. MTS-based (with MTS indicating 3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner salt) nonradioactive cell proliferation assay and Boyden chamber assay were performed to test their effect on tumor cell proliferation and migration. As expected, the conditioned medium of control HSCs promoted the proliferation and migration of HT-29 (Figure , A and B) and LLCs (Supplemental Figure 7) as compared with basal medium. Importantly, the conditioned medium of IQGAP1-knockdown HSCs exhibited a greater stimulatory effect on tumor cells than that of control HSCs (Figure , A and B, and Supplemental Figure 7). As detected by DAPI staining and WB for PARP cleavage, an early marker of cell apoptosis, these conditioned media protected MC38 cells from apoptosis in cell suspension culture and anoikis assays, and the conditioned media of IQGAP1-knockdown HSCs conferred a greater protection to MC38 cells (Figure C). These data support that IQGAP1 deficiency in activated HSCs may confer a greater stimulatory effect on the growth and survival of tumor cells through the release of soluble factors.
IQGAP1-knockdown HSCs promote the proliferation, migration, and survival of tumor cells.
Next, we isolated mRNAs from control and IQGAP1-knockdown HSCs for real-time quantitative RT-PCR analyses for paracrine cellular growth and motility factors, including TGF-β1, PDGF ligands, SDF-1/CXCL12, and HGF. Although the mRNA levels of TGF-β1 and PDGF ligands were not changed by IQGAP1 knockdown, the transcripts of SDF-1/CXCL12 and HGF were significantly increased by IQGAP1 knockdown in HSCs (Figure D). This finding is very interesting, since both SDF-1/CXCL12 and HGF play a central role in tumor metastasis and angiogenesis (47
) and SDF-1/CXCL12 has been identified as a chemokine that regulates organ-specific metastasis in various cancers (49
IQGAP1 in the myofibroblasts of patient colorectal liver metastases is downregulated.
Double IF for IQGAP1 and α-SMA was performed on liver biopsies of patients with colorectal cancers to determine IQGAP1 expression status in the stroma of established liver metastases. Liver biopsies of 29 colorectal cancer patients were obtained from a Mayo Clinic tissue collection. This patient cohort was 55% male and 45% female, and all were clinically diagnosed with metastatic colorectal cancer (Supplemental Table 1). The age of patients was from 32 to 90 years old, with a median of 63 years old. Their primary colon cancers originated from different colonic sites including ascending, transverse, descending, sigmoid colon, and rectum. After double IF for α-SMA and IQGAP1, IQGAP1 IF intensity in the myofibroblasts of the liver metastases was quantitated and compared with that in the myofibroblasts of matched control liver (Figure , A–C, and Supplemental Table 1). Out of 29 patients analyzed, 24 patients displayed varying degrees of reduction of IQGAP1 protein in the myofibroblasts of their liver metastases as compared with IQGAP1 expression levels observed in activated HSCs and portal myofibroblasts of the adjacent nontumorous control liver (Figure , A and C). This reduction was statistically significant in this cohort as detected by Student’s t test (P < 0.01) (Figure B).
IQGAP1 in the myofibroblasts of human colorectal liver metastases is downregulated.
Since metastatic growth in the liver is largely dependent on the communication between tumor cells and the hepatic tumor microenvironment, we next tested the hypothesis that when tumor cells intermingled with HSCs in the liver, tumor-derived factors might act on HSCs to reduce IQGAP1 expression of HSCs. To this end, conditioned medium of HT-29, MC38, and CT26 colorectal cancer cells were used to treat HSCs. As detected by WB, each conditioned medium tested indeed moderately reduced the IQGAP1 level of HSCs as compared with basal culture medium (Figure D). Furthermore, TGF-β1 (5 ng/ml) recapitulated the effect of the conditioned medium (Figure D), while PDGF-BB (20 ng/ml) did not (Supplemental Figure 8). Interestingly, IQGAP1 does not couple with TβRII for degradation after TGF-β1 stimulation, as shown in Figure A and Supplemental Figure 4B, indicating that IQGAP1 is downregulated by TGF-β1 through an alternative mechanism. Thus, that tumor-derived factors induced downregulation of IQGAP1 in the tumor-associated myofibroblasts may be important for the initiation and growth characteristics of colorectal liver metastasis in patients.