CRM197 reverses paclitaxel resistance by inhibiting the NAC‐1/Gadd45 pathway in paclitaxel‐resistant ovarian cancer cells

Abstract Heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF) is a new promising target for the treatment of ovarian cancer. Our previous study showed that cross‐reacting material 197 (CRM197), a specific HB‐EGF inhibitor, significantly reverses resistance against paclitaxel in paclitaxel‐resistant ovarian cancer cells. However, the mechanism of the effect of CRM197 on the reversion of paclitaxel resistance was unclear. In this study, in vitro and in vivo data suggested that CRM197 treatment sensitized paclitaxel‐resistant ovarian cancer cells to paclitaxel, at least in part, via nucleus accumbens‐1 (NAC‐1) and its downstream pathway, DNA damage‐inducible 45‐γ interacting protein (Gadd45gip1)/growth arrest and DNA damage‐inducible 45 (Gadd45), in A2780/Taxol and SKOV3/Taxol cells. The results also showed that CRM197 activated the proapoptotic JNK/p38MAPK pathway to enhance caspase‐3 activity and apoptosis by downregulation of the NAC‐1/Gadd45gip1/Gadd45 pathway, leading to reversion of paclitaxel resistance in A2780/Taxol and SKOV3/Taxol cells. This study provides the first mechanism through which CRM197 significantly reverses resistance against paclitaxel by modulating the NAC‐1/Gadd45gip1/Gadd45 pathway in paclitaxel‐resistant ovarian cancer cells, and the mechanism of HB‐EGF inhibition as a novel therapeutic strategy for patients with paclitaxel‐resistant ovarian cancer.


| INTRODUCTION
Currently, ovarian cancer is the most lethal gynecological malignancy. Paclitaxel has been widely used as a frontline therapeutic agent for ovarian cancer. 1 However, nearly all patients with ovarian cancer, who initially respond to paclitaxel, relapse and become refractory to chemotherapy. Moreover, therapies developed over the last 30 years have not improved survival rates. Patients who relapse or do not initially respond to traditional chemotherapy are thought to have drug-resistant cancer cells, resulting in cancer relapse and lethality. 2 To develop a novel effective therapy to restore the chemosensitivity of patients with ovarian cancer, further understanding of the molecules and mechanisms leading to paclitaxel resistance of ovarian cancer is needed.
Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is one of the seven ligands of the epidermal growth factor receptor (EGFR) and the primary EGFR ligand altered in ovarian cancer. 3,4 HB-EGF plays a critical role in proliferation, angiogenesis, and metastasis of ovarian | 6427 TANG eT Al.
cancer, making it a putative therapeutic target for ovarian cancer. Cross-reacting material 197 (CRM197), an HB-EGF inhibitor, is a nontoxic mutant of diphtheria toxin, which shares immunological properties with the native molecule. 5,6 CRM197 binds to the soluble form of HB-EGF as well as pro-HB-EGF and blocks mitogenic activity by inhibiting the binding between EGFR and HB-EGF. 7 Although CRM197 does not inhibit the mitogenic activity of other EGFR ligands, it is a specific inhibitor of HB-EGF and the only known inhibitor that can be used for cancer therapy in mice and humans. 8 Our previous in vitro and in vivo study demonstrated that CRM197 significantly reverses resistance against paclitaxel in paclitaxel-resistant ovarian cancer cells. 9 However, the mechanism of the reversion of paclitaxel resistance by CRM197 was unclear. In this study, we investigated the mechanism of CRM197 in alleviating paclitaxel resistance. First, we investigated the role of nucleus accumbens-1 (NAC-1) and its downstream pathway, growth arrest and DNA damage-inducible 45-γ interacting protein (Gadd45gip1)/ growth arrest and DNA damage-inducible 45 (Gadd45), in CRM197-mediated reversal of paclitaxel resistance in ovarian cancer cells. Second, we further investigated regulation of the MAPK pathway by the NAC-1/Gadd45gip1/Gadd45 pathway following treatment of ovarian cancer cells with CRM197. Third, we examined the effect of activation of the MAPK pathway by CRM197 on the caspase-3 activity and cell viability.

| Cell culture and CRM197 treatment
Human parental ovarian cancer cell lines (A2780 and SKOV3) and paclitaxel-resistant cell lines (A2780/Taxol and SKOV3/Taxol) were generously provided by Dr Lan Xiao (Department of Gynecology and Obstetrics, Third Affiliated Hospital of Sun Yan-sen University, Guangzhou, China) and cultured as described previously. 10 CRM197 (Sigma-Aldrich) treatment of parental and paclitaxel-resistant ovarian cancer cells was performed as described previously. 10

| Caspase-3 activity assay
The caspase-3 activity assay was performed using a colorimetric activity assay kit (R&D Systems), according to the manufacturer's instructions. Cells cultured with or without CRM197 were lysed, and total protein was measured by the Bradford assay. Samples were then analyzed for caspase-3 activity by Ac-DEVD as described previously. 11 Absorbance was measured at 405 nm in the ELX800 plate reader.

| Western blot analysis
Whole cell lysates (50 mg protein) were used for western blotting as described previously. 12 The proteins were separated by SDS-PAGE and then transferred to nitrocellulose membranes. The blot was probed with an anti-NAC-1 monoclonal antibody (Abcam PLC) at a 1:100 dilution, anti-Gadd45gip1 polyclonal antibody (Abcam PLC) at a 1:100 dilution, anti-p38 MAPK or -JNK monoclonal antibodies (Santa Cruz), and anti-phospho-p38 MAPK or anti-phospho JNK polyclonal antibodies (Abcam PLC). Antibody binding was detected using an enhanced chemiluminescence detection reagent (Amersham Biosciences), according to the manufacturer's protocol.

| NAC-1 short hairpin RNA
(shRNA) and Gadd45gip1 small interfering RNA (siRNA) transfections shRNA against NAC-1 and siRNA against Gadd45gip1 were purchased from Dharmacon Inc. Cells were grown to 80% confluence, and then NAC-1 shRNA, Gadd45gip1 siRNA, or scramble control siRNA were transfected into cells with or without CRM197 treatment using OligofectAMINE 2000 (Invitrogen) as described previously. 11 Western blotting was performed to examine the silenced protein levels after incubation for 48 hours.

| Xenografts
Mice were randomly divided into A2780, A2780/Taxol, and NAC-1 shRNA A2780/Taxol groups (eight mice/ group). PBS (200 μL) containing 1 × 10 7 A2780, A2780/ Taxol, or NAC-1 shRNA1 A2780/Taxol cells was subcutaneously injected into female BALB/c nude mice (Vital River Laboratory). The tumor volume was calculated by the formula 0.5 × width 2 × length. After the mean tumor size reached 100 mm 3 , A2780 and A2780/Taxol groups were randomly subdivided into CRM197 and control groups (four mice/group). After 4 weeks of CRM197 treatment, CRM197 dissolved in 200 μL PBS (1 mg/wk) was injected intraperitoneally into CRM197 groups (five mice/group) each week. The control groups (four mice/group) were injected intraperitoneally with 200 μL PBS for 4 weeks. Then, the mice were sacrificed and their tumors were subjected to immunohistochemical staining. All animal procedures were approved by the Committee on the Ethics of Harbin Medical University and complied with the Guidelines for the Care and Use of Laboratory Animals of Harbin Medical University.

| In vivo imaging
To visualize intraperitoneal tumors in mice, 100 mg/mL CRM197 was injected intraperitoneally, and macroscopic in vivo fluorescence and luminescence imaging was carried out using an IVIS system. A fresh solution of D-luciferin (OZ Biosciences) was prepared at 15 mg/mL in DPBS. For in vivo imaging, 48 hours after administration of the photosensitizer, the mice were injected intraperitoneally with 150 mg D-luciferin/kg body weight at 10-15 minutes before imaging. 14,15 Optical images obtained by the IVIS were analyzed with Living Image Software.

| Statistical analysis
Data are presented as means ± SD of three independent experiments. The Student's t-test was used for statistical analyses (Sigmastat v. 3.5 software). A P-value of <.05 was considered as significant.

| CRM197 enhances caspase-3 activity via the JNK/p38MAPK pathway
EGFR is the critical signal transducer linking HB-EGF to MAPK cascades. 16 Our previous study showed that HB-EGF inhibitor CRM197 significantly suppresses the expression of EGFR in ovarian cancer cells. 9 To determine whether the EGFR/JNK/p38MAPK pathway was affected by CRM197 treatment in ovarian cancer cells, we examined EGFR expression and JNK/p38MAPK pathway activity in ovarian cancer cells following CRM197 treatment. CRM197 significantly induced downregulation of EGFR expression ( Figure  1A) and activation of JNK/p38MAPK ( Figure 1B) in parental (A2780 and SKOV3) and paclitaxel-resistant (A2780/Taxol and SKOV3/Taxol) ovarian cancer cells (both P < .01), suggesting that CRM197 treatment activates the JNK/p38MAPK pathway by inhibiting EGFR expression. The increased expression pf EGFR and activation of JNK and p38MAPK in A2780/Taxol and SKOV3/Taxol cells were much higher than those in A2780 and SKOV3 cells, suggesting that activation of JNK and p38 MAPK is related to paclitaxel resistance.

NAC-1 expression
NAC-1, a tumor recurrence-associated gene, has important roles in cell survival and apoptosis by modulating apoptosis-related pathways. 17,18 However, the role of NAC-1 in CRM197-mediated reversal of paclitaxel resistance remains largely unclear. To elucidate the mechanism of CRM197 in reversion of paclitaxel resistance by regulation of the proapoptotic pathway, we first investigated the relationship between NAC-1 and CRM197. We measured NAC-1 expression after CRM197 treatment of parental (A2780 and SKOV3) and paclitaxel-resistant (A2780/Taxol and SKOV3/Taxol) ovarian cancer cells. The expression levels of NAC-1 mRNA ( Figure 2A) and protein ( Figure 2B) in A2780/Taxol and SKOV3/Taxol cells were significantly higher than those in A2780 and SKOV3 cells (P < .01), suggesting that NAC-1 contributes to paclitaxel resistance of ovarian cancer cells. We found that CRM197 significantly downregulated the expression of NAC-1 mRNA ( Figure 2A) and protein ( Figure 2B) in parental (A2780 and SKOV3) and paclitaxel-resistant (A2780/Taxol and SKOV3/ Taxol) cells (A2780 and SKOV3 cells, P < .01; A2780/Taxol and SKOV3/Taxol cells, P < .001).
To confirm the results, we established A2780 and A2780/ Taxol xenografted mice. Two weeks after subcutaneous injection, enhanced tumorigenicity was observed in paclitaxel-resistant ovarian tumors. During 4 weeks of CRM197 treatment, the tumor mass was measured every week, and then the mice were sacrificed and their tumors were collected for weight measurement. We examined NAC-1 protein expression by immunohistochemistry ( Figure 2C), and the tumor volume ( Figure 2D) and tumor weight ( Figure 2E) in A2780 and A2780/Taxol xenografts following CRM197 treatment. The staining intensity of NAC-1 in CRM197-untreated A2780/Taxol tumors was 3+, whereas the staining intensity of NAC-1 in CRM197-treated A2780/Taxol tumors was 1+. The staining intensity of NAC-1 in CRM197-untreated A2780 tumors was 2+, whereas the staining intensity of NAC-1 in CRM197-treated A2780 tumors was close to 0. Similar to the in vitro data, CRM197 effectively suppressed NAC-1 expression and the formation of A2780/Taxol tumors compared with A2780 tumors in vivo. A2780/Taxol cells, which expressed higher levels of NAC-1, formed larger tumors than A2780 cells.

| NAC-1 silencing enhances the effect of CRM197 on tumor growth
To confirm the role of NAC-1 in the effects of CRM197, we transiently transfected A2780/Taxol cells with shRNA against NAC-1 (NAC-1 shRNA1, shRNA2, and shRNA3) or control shRNA and examined their silencing effect by western blotting ( Figure 3A). Then, the NAC-1 expression level in response to CRM197 treatment was analyzed by western blotting in A2780/Taxol cells transfected with NAC-1 shRNA or control shRNA. Both CRM197 treatment and NAC-1 shRNA transfection (NAC-1 shRNA1, shRNA2, and shRNA3) significantly inhibited NAC-1 expression compared with the control shRNA ( Figure 3B, all P < .01). These data suggested that NAC-1 contributed to CRM197-mediated reversal of paclitaxel resistance. We next established A2780/Taxol NAC-1 shRNA (NAC-1 shRNA1) xenografted mice. First, we examined NAC-1 protein expression in xenografts following CRM197 treatment by immunohistochemical staining ( Figure 3C). Then, we detected the luminescent signals by fluorescence imaging ( Figure 3D) and measured the tumor size ( Figure 3E,F) and tumor weight ( Figure 3G) of control shRNA and NAC-1 shRNA A2780/Taxol xenografts following CRM197 treatment. The staining intensity of NAC-1 in CRM197-untreated control shRNA A2780/Taxol tumors was 3+, whereas the staining intensity of NAC-1 in CRM197-treated control shRNA A2780/Taxol tumors was 1+. The staining intensity of NAC-1 in CRM197-untreated NAC-1 shRNA A2780/Taxol tumors was 2+, whereas the immunointensity of NAC-1 in CRM197-treated NAC-1 shRNA A2780/Taxol tumors was close to 0. Similar to the in vitro data, the in vivo results showed that inhibition of NAC-1 expression by shRNA enhances the reversion of paclitaxel resistance by CRM197, suggesting that CRM197 significantly reverses resistance against paclitaxel by inhibiting NAC-1 expression in ovarian cancer cells.

| NAC-1 overexpression or silencing affects CRM197-mediated cell proliferation and caspase-3 activity
The above findings suggest that CRM197 not only mediates the proapoptotic JNK/p38MAPK pathway, but also downregulates NAC-1 expression. To investigate whether CRM197 activates the proapoptotic JNK/p38MAPK pathway by regulating NAC-1, we used two independent but complementary approaches. First, we ectopically expressed NAC-1 tagged with V5 (V5-1, V5-2, and V5-3) in normal epithelial cell line RK3E with undetectable NAC-1 expression and analyzed the change in activation of the JNK/p38MAPK pathway ( Figure 4A,B, both P < .05). RK3E cells are insensitive to taxol, and we found low expression of NAC-1 in RK3E cells ( Figure 4A). Activation of the JNK/p38MAPK pathway was significantly reduced in RK3E cells transfected with the V5-NAC-1 vector compared with control vector-transfected cells ( Figure 4B). Second, we observed a decrease in the NAC-1 expression level with a concomitant increase in activation of the JNK/p38MAPK pathway in A2780/Taxol cells ( Figure  4B, P < .05). These data revealed that CRM197 activates the JNK/p38MAPK pathway by downregulation of NAC-1 expression.

T A B L E 2
Further investigation of the relationships among Gadd45gip1, Gadd45γ, and P38/JNK was performed in A2780/Taxol cells. We knocked down Gadd45gip1 in A2780/ Taxol cells using Gadd45gip1 shRNA (Gadd45gip1 shRNA1, shRNA2, and shRNA3; Figure 5D, P < .01) and observed a decrease in the Gadd45gip1 expression level with concomitant decreases in the levels of both Gadd45γ and p38MAPK/ JNK ( Figure 5E, P < .01), suggesting that expression of Gadd45γ and p38MAPK/JNK is dependent on Gadd45gip1 expression. Taken together, these data demonstrated that CRM197 markedly downregulates the NAC-1 expression by upregulating the Gadd45gip1/Gadd45γ/p38MAPK/JNK pathway in A2780/Taxol cells ( Figure 6).

| DISCUSSION
CRM197 is currently the only known HB-EGF inhibitor that can be used for cancer therapies in mice and humans. We and others have shown that CRM197 is a promising F I G U R E 5 CRM197 decreases NAC-1 expression by upregulation of the Gadd45gip1/Gadd45γ pathway in paclitaxel-resistant ovarian cancer cells. A, CRM197 increased expression of Gadd45gip1 and Gadd45γ in parental (A2780 and SKOV3) and paclitaxel-resistant (A2780/Taxol and SKOV3/Taxol) ovarian cancer cells. B, Expression levels of Gadd45gip1 and Gadd45γ were detected in A2780/Taxol cells treated with NAC-1 shRNA (NAC-1 shRNA1) or control shRNA by western blotting (P < .01). C, Expression levels of Gadd45gip1 and Gadd45γ were detected in RK3E cells transfected with the V5-NAC-1 vector (V5-1) or control vector by western blotting (P < .01). D, A2780/Taxol cells were transiently transfected with Gadd45gip1 siRNA (Gadd45gip1 siRNA1, siRNA2, and siRNA3) or the scramble empty vector (Empty), or treated with PBS (Con, control). Western blotting for Gadd45gip1 was performed to detect the inhibition efficiency of siRNA. E, Expression levels of Gadd45γ and activation of p38MAPK/JNK were detected in A2780/Taxol cells transfected with Gadd45gip1 siRNA (Gadd45gip1 siRNA1, siRNA2, and siRNA3) or control siRNA