RacGAP1 Is a Novel Downstream Effector of E2F7-Dependent Resistance to Doxorubicin and Is Prognostic for Overall Survival in Squamous Cell Carcinoma

RacGAP1 is a novel downstream effector of E2F7

We generated E2f7 knockdown (KD) murine KCs via adenovirus-mediated Cre deletion of floxed sequences in primary KCs isolated from E2f7-floxed mice (15). KD KCs were treated for 48 hours with increasing concentrations of doxorubicin (0–1 μmol/L), etoposide (0–100 μmol/L), and cisplatin (0–20 μmol/L). Figures 1A–C show that E2F7 deficiency sensitizes KCs to doxorubicin, modestly to cisplatin, but not at all to etoposide. These data suggest that E2F7-mediated doxorubicin resistance is not attributable to topoisomerase inhibition because etoposide sensitivity was not modified by E2F7. Moreover, pan-caspase inhibition significantly protected E2F7-deficient cells from doxorubicin-induced cytotoxicity (Fig. 1D), indicating that apoptotic pathways are being activated.

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Figure 1.

Cytotoxic responses to doxorubicin in E2F7-deficient murine KCs. E2F7-floxed KCs were incubated with (squares) or without (circles) Ad-Cre-GFP for 48 hours and then incubated with varying doses of doxorubicin (A), etoposide (B), or cisplatin (C). Viability (absorbance 490 nm) was assessed 48 hours after treatment and is expressed as the mean ± SEM obtained from triplicate determinations of three independent experiments. D, Ad-Cre-GFP–uninfected E2F7-floxed or Ad-Cre-GFP–infected E2F7-deficient proliferative KCs were treated with 1 μmol/L doxorubicin in the presence or absence of ZVAD-fmk and viability determined 48 hours later. Viability is plotted as a percentage of doxorubicin-treated uninfected E2F7-floxed MEKs and represents the mean ± SEM obtained from triplicate determinations of three independent experiments. E, HEK, Detroit562, KJDSV40, SCC15, and SCC25 cells were treated with doxorubicin for 48 hours and viability plotted as percentage of untreated cells. Inset, estimated IC₅₀ values for doxorubicin in HEK, KJDSV40, and SCC25 cells determined by nonlinear regression analysis in Prism. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

We undertook a screen of doxorubicin sensitivity in HEKs and 4 SCC cell lines. The KJDSV40 cell line exhibited the highest sensitivity to doxorubicin (IC₅₀ of 0.082 μmol/L; Fig. 1E), whereas SCC25 cells displayed the least sensitivity (IC₅₀ of 0.55 μmol/L; Fig. 1E) and HEKs displayed intermediate sensitivity (IC₅₀ of 0.29 μmol/L; Fig. 1E). We have previously shown that the insensitive SCC25 cell line expresses high levels of E2F7, whereas the sensitive KJDSV40 cell line expresses low levels of E2F7 (15). Based on these data, we selected the sensitive KJDSV40 cell line and the insensitive SCC25 cell line for transcriptomic profiling.

Specifically, we generated a list of genes that were poorly expressed in KJDSV40 cells and highly expressed in SCC25 (Fig. 2A). We also generated a second list of genes that were differentially regulated in SCC25 cells in which E2F7 had been silenced with siRNA (Fig. 2A). We then used these two lists to identify those transcripts (referred to as List A; Supplementary Table S1) that displayed E2F7-dependent expression between the SCC25 cell lines (Fig. 2A). We also generated an additional list of transcripts for SCC25 cells or SCC25 cells in which E2F7 is silenced by siRNAs that have been treated with 1 μmol/L of doxorubicin. The transcripts that were found to be E2F7-dependant in the context of doxorubicin-treated SCC25 cells were then referred to as List B (Fig. 2A; Supplementary Table S1). By combining Lists A and B, we identified RacGAP1 as the most differentially overexpressed genes with a B value of 17 (Supplementary Table S1).

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Figure 2.

RacGAP1 is a downstream effector of E2F7. A, summary of the strategy used to identify E2F7-dependent transcripts that associate with doxorubicin resistance in SCC cells. B and C, RacGAP1 mRNA and protein levels were determined in KJDSV40 and SCC25 cells by qRT-PCR and immunoblotting, respectively. D and E, the expression of RacGAP1 transcripts and protein was determined by qRT-PCR and immunoblotting, respectively, of extracts derived from SCC25 and SCC25 cells in which E2F7 was silenced with siRNA for 48 hours. F and G, RNA was extracted from KJDSV40 and KJDSV40 cells in which E2F7 was overexpressed from an expression plasmid. The expression of RacGAP1 transcripts and protein was determined 48 hours after transfection by qRT-PCR and immunoblotting, respectively. H, to quantitate E2F7 binding to the RacGAP1 promoter, ChIPs were performed using an E2F7 antibody or nonimmune IgG as control in HEK, KJDSV40, and SCC25 cells. Each ChIP and qRT-PCR was repeated 2 or 3 times, respectively. Data, mean ± SEM of duplicate determinants normalized for expression of the housekeeping gene TBP for B, D, and F; n = 3. β-Actin is provided as a loading control for C, E, and G. Western blot figure is representative of three independent experiments. **, P < 0.01;****, P < 0.0001.

RacGAP1 (also known as MgcRacGAP and CYK4) is an evolutionarily conserved GTPase activating protein (GAP) that displays activity toward the Rho family of GTPases. The Rho family of GTPases is a subfamily of the Ras superfamily and consists of small signaling G proteins: Rho (A, B, and C isoforms), Rac (1,2,3 isoforms and RhoG), and Cdc42 (Cdc42, Tc10, TCL, Chp/Wrch-2, and Wrch-1; ref. 24), which function as molecular switches between a GTP-loaded “ON” and a GDP-loaded “OFF” state (24). Thus, RacGAP1 has the potential to regulate a diverse array of cellular functions through its central role as a regulator of the activation state of the Rho family of GTPases. In particular, RacGAP1 is known to play important roles in the completion of cytokinesis (25), cell transformation, motility, migration, and metastasis (26–29). RacGAP1 is also involved in IL6-induced macrophage differentiation (30) and nuclear transport of STAT3/5 transcription factors (31). However, a role for RacGAP1 in SCC or doxorubicin sensitivity has not been shown previously.

Quantitative RT-PCR and Western blotting were used to confirm that RacGAP1 was more highly expressed in SCC25 (doxorubicin insensitive) cells than in KJDSV40 (doxorubicin sensitive) cells (Fig. 2B and C). Similarly, we showed that knockdown of E2F7 by siRNA in SCC25 cells caused a reduction in RacGAP1 mRNA (Fig. 2D) and protein level (Fig. 2E). Conversely, overexpression of E2F7 in KJDSV40 cells resulted in elevated levels of RacGAP1 transcript (Fig. 2F) as well as RacGAP1 protein (Fig. 2G). These data suggest that RacGAP1 is a downstream target of E2F7 in SCC cells. Supporting this, ChIP analysis of E2F7 binding showed that E2F7 could bind the RacGAP1 promoter, suggesting that RacGAP1 is a direct transcriptional target of E2F7 (Fig. 2H). This is the first report to show that RacGAP1 is a downstream effector of E2F7.

RacGAP1expression is elevated in SCCs

We examined RacGAP1 expression levels by immunohistochemistry using a TMA consisting of 35 paired normal, primary tumor, and matched metastasis from HNSCC patients treated at the Princess Alexandra Hospital (PAH). The TMAs were stained for E2F7 and RacGAP1 protein expression and scored blinded by two pathologists. All matched adjacent “normal” epithelia demonstrated either negative or weak staining for RacGAP1, which was predominantly nuclear in location (Fig. 3A). Conversely, moderate to high levels of RacGAP1 expression were consistently recorded for the primary tumor (Fig. 3B) and its matched lymph node metastasis (Fig. 3C). The tumor epithelial cells showed nuclear and cytosolic expression for RacGAP1. RacGAP1 was significantly overexpressed in 73% of primary and metastatic human SCCs compared with matched adjacent normal tissue (P < 0.0001; Fig. 3D). In addition, our analyses showed that E2F7 expression is significantly upregulated in HNSCC compared with matched adjacent normal tissue (P < 0.0001; Fig. 3D). The Kaplan–Meier analysis revealed an inverse relationship between RacGAP1 expression levels and progression-free survival (PFS) of HNSCC patients studied over a period of 42 months whose samples were arrayed on the TMA (Fig. 3E). These data show, for the first time, that RacGAP1 is overexpressed in HNSCC and is associated with a poorer PFS.

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Figure 3.

Expression of RacGAP1 in HNSCC and adjacent normal tissue and its association with PFS. Representative images of adjacent normal tissue (A) and HNSCC specimens stained for RacGAP1 (B and C). D, quantitation of E2F7 and RacGAP1 staining intensities in matched samples of primary tumor, normal squamous epithelium, and lymph node metastases (n = 35). Tissue sections were scored using a modified quickscore method to determine the percentage of cells stained (0%–100%) and the intensity of staining (1+ to 3+). E, Kaplan–Meier analysis of PFS stratified by RacGAP1 expression in the HNSCC patient cohort. ns, not significant, ****, P < 0.0001.

RacGAP1 expression/activity determines sensitivity to doxorubicin

We examined the effect of shRNA-mediated knockdown or RacGAP1 overexpression on sensitivity to doxorubicin. RacGAP1 gene silencing was achieved using 4 different constructs of which shRNA.3 displayed the greatest knockdown in RacGAP1 protein level (Fig. 4A). For subsequent experiments, the shRNA complex shRNA.3 was employed. Consistent with previous reports (32), RacGAP1 shRNA-transfected SCC25 cells displayed significant reductions in proliferation (Fig. 4B), colony-forming efficiency in vitro (Fig. 4C), and induced a modest increase in cleaved PARP1 (Fig. 4D) compared with control vector–transfected cells. Finally, silencing RacGAP1 significantly enhanced the sensitivity of SCC25 cells to doxorubicin (Fig. 4E). Conversely, overexpression of RacGAP1 in sensitive KJDSV40 cells resulted in increases in RacGAP1 protein level (Fig. 4F), and reduced sensitivity to doxorubicin compared with vector control (Fig. 4G). Combined, these data indicate that RacGAP1 can promote proliferation and inhibit doxorubicin-induced cell death in SCCs.

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Figure 4.

The sensitivity to doxorubicin is mediated via an E2F7/RacGAP1 axis in SCC. A, SCC25 cells were transfected with 4 different constructs coding for shRNAs directed against RacGAP1. After 48 hours, RacGAP1 protein expression was determined by immunoblotting. β-Actin is provided as a loading control. SCC25 cells were transfected with the RacGAP1shRNA.3 or a scrambled shRNA construct. After 48 hours, BrdUrd incorporation (B) and CFE (C) were determined. Data are expressed as a percentage of that observed for control shRNA. D, cleavage of PARP was determined by immunoblotting extracts of RacGAP1shRNA and control shRNA–transfected SCC25 cells. E, RacGAP1shRNA and control shRNA–transfected SCC25 cells were treated with doxorubicin (1 μmol/L) for 48 hours and viability estimated by trypan blue exclusion. Viability is plotted as percentage control (untreated). F, KJDSV40 cells were transfected with RacGAP1 expression plasmid or a noncoding empty vector. After 48 hours, RacGAP1 protein expression was determined by immunoblotting. β-Actin is a loading control. G, RacGAP1 expression plasmid or empty vector–transfected KJDSV40 cells were treated with doxorubicin (1 μmol/L) for 48 hours and viability estimated by trypan blue exclusion. Viability was expressed as percentage control (untreated). Western blot figures are representative of three independent experiments. All quantitative data presented as mean ± SEM. For viability, values represent triplicate determinations of three independent experiments; for CFE, expression values represent duplicate determinations from at least three independent experiments; for BrdUrd, values represent triplicate determinations from 4 independent experiments. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

RacGAP1 differentially regulates the GTP-loaded state of RhoA and Rac1 in SCC cells

We examined whether the overexpression of RacGAP1 in the SCC cell lines was reflected in alterations of the GTP loading (activation status) of the model targets RhoA and Rac1. Specifically, RhoA GTP loading was constitutively higher in KJDSV40 cells, which express very low levels of E2F7 and RacGAP1, compared with SCC25 cells which express high levels of E2F7 and RacGAP1 (Fig. 5A). In contrast, GTP loading of Rac1 was higher in SCC25 cells when compared with KJDSV40 cells (Fig. 5B), and the GTP loading of Rac1 was significantly reduced in SCC25 cells following RacGAP1 knockdown (Fig. 5B). Finally, knockdown of RacGAP1 in SCC25 cells resulted in an increase in the GTP loading of RhoA (Fig. 5A). These data suggest that E2F/RacGAP1-dependent resistance to doxorubicin may be mediated by activated Rac1 or inactivated RhoA. To determine which, if any, of these possibilities may apply, we coincubated SCC25 cells with doxorubicin +/– an established selective Rho/ROCK1 inhibitor (10 μmol/L Y-27632; ref. 33), an established Rac1/Cdc42 inhibitor (10 μmol/L AZA1; ref. 34), or a Rac1-selective inhibitor (25 μmol/L NSC23766; ref. 35) for 48 hours and established viability (Fig. 5C). These experiments showed that doxorubicin resistance was unaltered following incubation with a Rho/ROCK1 inhibitor and was moderately reduced following incubation with the Rac1/cdc42 inhibitor (Fig. 5C). In contrast, treatment of SCC25 cells with the Rac1-selective inhibitor significantly increased sensitivity of SCC25 cells to doxorubicin (Fig. 5C). These results indicate a number of important points. Firstly, RhoA appears to be a preferred substrate for RacGAP1 in SCC cells. This is reflected by the high level of RhoA-GTP loading compared with Rac1-GTP loading as well as the increase in GTP loading observed following RacGAP1 knockdown in the SCC25 cells. Secondly, although Rac1-GTP loading behavior is not indicative of it being a preferred substrate of RacGAP1, it is clear that alterations in RacGAP1 activity modify Rac1-GTP loading. Thirdly, use of a Rac1-selective inhibitor phenocopies the inhibition of doxorubicin resistance observed with RacGAP1 knockdown (Fig. 4E). Finally, the preference for RhoA by RacGAP1, in SCC cells, is consistent with a previous report showing that the conventional preference for Rac1 can be switched to RhoA following phosphorylation of the Serine 387 site of RacGAP1 by Aurora B kinase (36, 37).

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Figure 5.

Alterations in RacGAP1 activity modify Rac1-GTP loading, and RacGAP1 lies upstream of AKT. Rhotekin-binding domain (RBD; A) binding and p21 activated kinase I-binding domain (PAK-PBD; B) assays were performed on the extracts from KJDSV40, SCC25, and SCC25 cells in which RacGAP1 had been silenced by shRNA, as indicated. The amount of activated or total RhoA and Rac1 was detected by immunoblotting the RBD and PAK-PBD samples and the whole cell lysate with RhoA or Rac1 antibodies. To confirm equal input, the membrane was reprobed with an actin antibody. C, SCC cells were treated with increasing doses of doxorubicin in combination with 10 μmol/L AZA1 or 10 μmol/L NSC23766 or 25 μmol/L NSC23766 for 48 hours. Viability was then assessed and plotted as percentage doxorubicin only treated. SCC25 cells were transfected with the RacGAP1shRNA or a scrambled shRNA construct. D, after 48 hours p-ERK and total ERK protein levels and (E) p-AKT and total AKT protein levels were determined by immunblotting. F, p-AKT and total AKT protein levels are shown for KJDSV40 cells in which RacGAP1 was overexpressed. G, RacGAP1shRNA and control shRNA–transfected SCC25 cells were treated with BGT226 (300 nmol/L) for 48 hours and viability estimated by trypan blue exclusion and plotted as percentage control shRNA. H, SCC25 cells were transfected with Sphk1shRNA (left) and RacGAP1shRNA (right) as well as control shRNA. After 48 hours, RacGAP1 (left) and Sphk1 mRNA (right) levels were determined by qRT-PCR. I, SCC25 cells were transfected with RacGAP1 or Sphk1 shRNAs. Forty-eight hours after transfection, cells were treated with 1 μmol/L S1P for 24 hours. p-AKT and total AKT protein levels were then determined by immunoblotting. J, SCC25 and SCC25 in which RacGAP1 or Sphk1 had been silenced were treated with 1 μmol/L doxorubicin and 1 μmol/L S1P for 24 hours. Viability was then assessed and plotted as percentage doxorubicin-only treated. Western blot figures are representative of three independent experiments. All quantitative data presented as mean ± SEM obtained from triplicate determinations of three independent experiments for C, G, and J. Data are the mean ± SEM of duplicate determinants normalized for expression of the housekeeping gene TBP for H; n = 3. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

RacGAP1 modulates doxorubicin sensitivity via downstream activation of the PI3K/AKT pathway

There is an existing literature showing that the PI3K/AKT pathway is an important component of RacGAP1 signaling (38). However, whether PI3K/AKT signaling lies upstream or downstream of the Rho family of GTPases remains less clear and appears to be context-specific (38). Dysregulation of the PI3K/AKT pathway is a common event in HNSCC, which can be attributed to multiple factors, such as mutations, amplifications, and signal-induced activation of the pathway (39). For example, we recently showed that E2F7 overexpression or knockdown caused an increase and decrease in p-AKT levels in SCC cells respectively (15). Since RacGAP1 is a downstream effector of E2F7 in SCC cells, we examined whether RacGAP1 could modify the PI3K/AKT signaling pathway in SCC cells. In the first instance, we noted that knockdown of RacGAP1 in SCC25 cells had no impact on the activation status of the ERK pathway (Fig. 5D). In contrast, RacGAP1 knockdown in SCC25 cells significantly reduced the level of p-AKT (Fig. 5E), whereas RacGAP1 overexpression in KJDSV40 cells increased p-AKT levels (Fig. 5F). We had previously shown that the PI3K/mTOR inhibitor, BGT226, was able to ablate AKT signaling and induce apoptosis in SCC cell lines (16). We compared the sensitivity of SCC25 cells with BGT226 in SCC25 cells or SCC25 cells in which RacGAP1 was knocked down. Figure 5G indicates that knockdown of RacGAP1 is able to reduce SCC25 cell viability to 70% that of control cells. Similarly, inhibition of PI3K activity using a dose of BGT226 known to induce maximal inhibition (16) reduced SCC25 cell viability to approximately 50% (Fig. 5G). Finally, exposure of RacGAP1-deficient SCC25 cells to BGT226 resulted in a further decrease in viability to below 20% (Fig. 5G). These data indicate that RacGAP1 participates in AKT-dependent and AKT-independent events.

We recently reported that E2F7 is able to directly activate the Sphk1/S1P axis in SCC cells, which induces doxorubicin resistance (15). It is also interesting to note that both the E2F7/RacGAP1 pathway identified in this study and the E2F7/Sphk1/S1P pathway (15) induced doxorubicin resistance and converged on the AKT pathway. Therefore, we examined whether the Sphk1 and RacGAP1 pathways may interact with one another. Figure 5H shows that knockdown of Sphk1 can induce loss of RacGAP1 mRNA, whereas knockdown of RacGAP1 induces loss of Sphk1 mRNA expression. Although the mechanism controlling this feedback is unknown, it is clear that targeted inhibition of either the RacGAP1 pathway or the Sphk1 pathway is likely to impact one another. To illustrate this point, knockdown of RacGAP1 or Sphk1 in SCC25 cells results in reduced p-AKT levels (Fig. 5I) and increased sensitivity to doxorubicin (Fig. 5J), which can be reversed by the addition of exogenous S1P (Fig. 5I and J).

RacGAP1 suppression enhances sensitivity of SCC25 to doxorubicin in vivo

SCC25 cells were generated to stably express either vector control or RacGAP1 shRNA and inoculated into NOD/SCID mice. When tumors were around 4 mm in diameter, mice were randomized into four groups and treated with vehicle, dimethyl sulfoxide (DMSO), or 0.5 mg/kg doxorubicin by i.p. injections twice per week. RacGAP1 knockdown was confirmed by Western blotting immediately before the inoculation of SCC25 cells (Fig. 6A). Treatment with doxorubicin was well tolerated by the NOD/SCID mice, and the body weights remained stable throughout the study (Fig. 6A). RacGAP1-deficient cells showed reduced tumor growth rate (Fig. 6B). Treatment of mice bearing vector control SCC25 tumors with/without 0.5 mg/kg doxorubicin had minimal effect on tumor growth rates (Fig. 6B). However, RacGAP1-deficient SCC25 tumors treated with doxorubicin started to regress by day 4 after treatment, which continued for a further 7 days at which time all mice were sacrificed due to the tumor burden in control mice. The subcutaneous tumors were excised, photographed, and examined histologically (Fig. 6C).

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Figure 6.

RacGAP1 suppression enhanced sensitivity of SCC25 to the cytotoxic actions of doxorubicin in vivo. A, on the day of subcutaneous injections, RacGAP1 deficiency was confirmed by immunoblotting using protein extracts from SCC25 cells in which RacGAP1 had stably silenced with shRNA. β-Actin is provided as a loading control. All animals were inoculated subcutaneously with 2 × 10⁶ SCC25 cells expressing vector alone (scrambled shRNA) or Sphk1 shRNA and tumors allowed to establish until they reached the indicated sizes. Established tumors were treated with vehicle or 0.5 mg/kg doxorubicin twice per week. A, animal weight was determined twice per week. B, tumor volumes were monitored twice weekly. The inset includes vehicle or 0.5 mg/kg doxorubicin-treated SCC tumors harboring RacGAP1shRNA. C, after 13 days of treatment, animals were sacrificed and tumors excised. Representative results from distinct groups are shown. +, tumors formed from control shRNA–transfected SCC25 cells; −, tumors formed from RacGAP1shRNA–transfected SCC25 cells. D, immunostaining for RacGAP1, PCNA, cleaved caspase-3, and p-AKT or normal Rabbit IgG as a negative control. Representative images of at least three independent tumors are shown for each group (Bar, 100 μm). Data presented as mean ± SEM of individual measurements from 6 mice per group. E, schematic model showing how disruption of the novel E2F/RacGAP1/Rac1/AKT pathway can lead to doxorubicin resistance in SCC.

Immunohistochemical examination of the excised tumors showed that knockdown of RacGAP1 was maintained throughout the study (Fig. 6D). Tumors from vehicle and doxorubicin-treated control mice stained strongly for PCNA, indicating a higher proportion of proliferating cells in control tumors compared with RacGAP1-deficient tumors (treated or untreated; Fig. 6D). In contrast, doxorubicin induced higher apoptosis indices in tumors derived from SCC25/RacGAP1shRNA than in the SCC25/vector control as estimated by immunostaining for cleaved caspase-3 (Fig. 6D). Ser473 p-AKT levels in RacGAP1-deficient cells were also decreased (Fig. 6D). Collectively, these results suggest that RacGAP1 contributes to the growth of HNSCC in vivo and that targeted inhibition of RacGAP1-overexpressing tumors may sensitize them to the cytotoxic actions of doxorubicin.