The Tumor-Suppressor Protein OPCML Potentiates Anti–EGFR- and Anti–HER2-Targeted Therapy in HER2-Positive Ovarian and Breast Cancer Elisa Zanini1, Louay S

Published OnlineFirst August 3, 2017; DOI: 10.1158/1535-7163.MCT-17-0081

Cancer Biology and Signal Transduction Molecular Cancer Therapeutics The Tumor-Suppressor Protein OPCML Potentiates Anti–EGFR- and Anti–HER2-Targeted Therapy in HER2-Positive Ovarian and Breast Cancer Elisa Zanini1, Louay S. Louis1, Jane Antony1, Evdoxia Karali1, Imoh S. Okon1,2, Arthur B. McKie1,3, Sebastian Vaughan1, Mona El-Bahrawy4, Justin Stebbing5, Chiara Recchi1, and Hani Gabra1,6

Abstract

Opioid-binding protein/cell adhesion molecule-like (OPCML) erlotinib, respectively, were developed to target these receptors, is a tumor-suppressor gene that is frequently inactivated in but resistance often occurs in relapsing cancers. Here we show ovarian cancer and many other cancers by somatic methylation. that, though OPCML interacts only with HER2 and not with We have previously shown that OPCML exerts its suppressor EGFR, the interaction of OPCML with HER2 disrupts the forma- function by negatively regulating a spectrum of receptor tyrosine tion of the HER2-EGFR heterodimer, and this translates into a kinases (RTK), such as ErbB2/HER2, FGFR1, and EphA2, thus better response to both lapatinib and erlotinib in HER2- attenuating their related downstream signaling. The physical expressing ovarian and breast cancer cell lines. Also, we show interaction of OPCML with this deﬁned group of RTKs is a that high OPCML expression is associated with better response to prerequisite for their downregulation. Overexpression/gene lapatinib therapy in breast cancer patients and better survival in ampliﬁcation of EGFR and HER2 is a frequent event in multiple HER2-overexpressing ovarian cancer patients, suggesting that cancers, including ovarian and breast cancers. Molecular thera- OPCML co-therapy could be a valuable sensitizing approach to peutics against EGFR/HER2 or EGFR only, such as lapatinib and RTK inhibitors. Mol Cancer Ther; 16(10); 2246–56. 2017 AACR.

Introduction 83% of epithelial ovarian cancers (EOC; ref. 2). OPCML methylation in EOC has also been confirmed by other studies (3, 4). Opioid-binding protein/cell adhesion molecule-like (OPCML) Furthermore, OPCML is inactivated in numerous other types of is a glycosyl phosphatidylinositol (GPI) anchored molecule cancers, such as brain, nasopharyngeal, esophageal, gastric, hepa- that is expressed in virtually all normal adult and fetal tissues tocellular, colorectal, breast and cervical cancers, as well as lym- (1). We first identified OPCML as a putative tumor-suppressor phomas (1, 4–6). In lung adenocarcinoma, OPCML is one of the gene (TSG) at chromosome 11q25, a site highlighted by frequent four most commonly methylated genes compared with normal loss of heterozygosity (LOH), and further demonstrated that lung tissue (7, 8). All these data indicate that OPCML expression is epigenetic silencing of the remaining OPCML allele occurred in very frequently lost across different types of cancers. Recently, we have defined that OPCML acts as a TSG by negatively regulating a set of receptor tyrosine kinases (RTK), including HER2, HER4, 1Ovarian Cancer Action Research Centre, Department of Surgery and Cancer, EphA2, FGFR1, and FGFR3 (9). In particular, OPCML binding to 2 Imperial College London, London, United Kingdom. Center for Molecular and the extracellular domain (ECD) of HER2 promotes HER2 ubiqui- Translational Medicine, Georgia State University, Atlanta, Georgia. 3Department tination and proteasomal degradation via clathrin-independent of Medical Genetics, University of Cambridge, Addenbrooke's Treatment Centre, Cambridge Biomedical Campus, Cambridge, United Kingdom. 4Department of endocytosis (9). Conversely, silencing of OPCML causes upregu- Histopathology, Imperial College London, London, United Kingdom. 5Depart- lation of this same repertoire of RTKs and increased signaling (9). ment of Surgery and Cancer, Imperial College London, London, United Kingdom. OPCML therefore induces a system level switch-off of a spectrum 6Clinical Discovery Unit, Early Clinical Development, AstraZeneca, Cambridge, of RTKs, which eventually leads to a strong decrease in phosphor- United Kingdom. ylation and activation of key downstream signaling molecules Note: Supplementary data for this article are available at Molecular Cancer such as ERK and AKT, thus reducing cell proliferation in vitro and Therapeutics Online (http://mct.aacrjournals.org/). in vivo (9). E. Zanini and L.S. Louis contributed equally to this article. HER2 is an RTK whose overexpression drives cell proliferation in Corresponding Authors: Hani Gabra, Imperial College London, Ovarian Cancer many human cancers. Studies have shown that ovarian tumors Action Research Centre, Hammersmith Hospital, Du Cane Road, London W12 with high malignant potential overexpress HER2 in 22% to 66% of 0NN, United Kingdom. Phone: 44–208–383–5828; Fax: 44–208–383–5830; cases (10), although recent consensus reduces the number of cases E-mail: [email protected]; and Chiara Recchi, Imperial College London, with genomic amplification of HER2 to 11% (11). In breast cancer, Ovarian Cancer Action Research Centre, Hammersmith Hospital, Du Cane Road, HER2 amplification is reported in 15% to 20% of cases (12). In London W12 0NN, United Kingdom; [email protected] comparison with the above, EGFR overexpression can be as high as doi: 10.1158/1535-7163.MCT-17-0081 28% and amplified in up to 20% of ovarian cancer patients (13) 2017 American Association for Cancer Research. and in 13% to 52% of triple-negative breast cancers (14).

2246 Mol Cancer Ther; 16(10) October 2017

OPCML Sensitizes HER2þ Cancers to Anti-HER2/EGFR Therapy

The concept of "oncogene addiction," by which the growth of used at 1 mg/mL as a selective antibiotic for SKOV3 cell lines, at some tumors relies heavily on the amplification of a single 0.1 mg/mL for OV90 cells and at 0.2 mg/mL for SKBR3. oncogene (15), as well as its downstream pathway, led to the COS-1 simian kidney fibroblast cells (CV-1) were maintained development of targeted therapies directed against HER2 and in DMEM, supplemented with 10% FCS, 2 mmol/L LG, 50 U/mL EGFR. Trastuzumab (a monoclonal antibody targeting HER2) Penicillin/Streptomycin. and lapatinib (a small-molecule inhibitor against HER2 and All cells were tested regularly to exclude mycoplasma infection. EGFR) have been used successfully in the treatment of metastatic COS-1 cells were tested and authenticated as described previously HER2-positive breast cancer along with chemotherapy, resulting (9). All the other cell lines were purchased from the ATCC in in increased response rate, prolonged progression-free survival January 2013. and extended overall survival compared with chemotherapy alone (16, 17). However, only 20% to 30% of HER2 positive Antibodies breast tumors respond to single HER2-targeted therapy (18) and Polyclonal goat anti-human OBCAM (OPCML) antibody was those that initially respond eventually develop recurrence due to purchased from R&D Systems. Total and phospho-HER2 drug resistance (19). Erlotinib (Tarceva, an EGFR-specific inhib- (Y1248), total and phospho-EGFR (Y1068), total and phos- itor) has been approved for the treatment of recurrent non–small pho-AKT (S473) antibodies were purchased from Cell Signaling cell lung cancer (NSCLC; ref. 20) with a specific EGFR mutation Technology. Total HER2 antibody (OP16L) used for Duolink was (21, 22). Despite the positive initial response, also these patients purchased from Merck Millipore. Calnexin antibody was pur- develop resistance after 10 to 13 months of treatment with chased from ENZO Life Sciences. erlotinib (23). Mechanisms of resistance to tyrosine kinase inhibitors (TKI) include the activation of alternative pathways Western blotting through RTK cross-talk (24–29) and mutations in the tyrosine Before collection, cells were washed in PBS. Whole-cell lysates kinase domain of the receptors (30–32). Several alternative were collected in RIPA buffer (Sigma-Aldrich) supplemented with approaches have been proposed to overcome drug resistance, protease inhibitor cocktail (Roche) and phosphatase inhibitor such as the concomitant use of lapatinib and trastuzumab upfront cocktail II (Calbiochem) at the manufacturers' recommended and the creation of new small-molecule inhibitors (33), and these concentration. Protein concentration was estimated using the are currently being evaluated. BCA assay (Pierce) according to the manufacturer's recommenda- BecausewehaveshownthattheexpressionofOPCMLisa tions. Lysates were incubated at 95 C for 5 minutes and then good prognostic factor for progression-free survival in ovarian separated into 10% sodium dodecyl sulphate polyacrylamide gel and breast cancer patients and that it induces HER2 down- electrophoresis (SDS-PAGE). Gels were then transferred onto regulation in ovarian cancer cells (9), OPCML restoration nitrocellulose membranes (VWR) that were then blocked with could represent a novel approach to reduce the level of 5% milk/TBST. Membranes were incubated with primary anti- activity of this oncogene and thereby sensitize cancer cells to bodies in 5% BSA/TBST or 5% milk/TBST at 4 C overnight. HRP- anti-HER2 therapy. Furthermore, as HER2 is the preferred conjugated secondary antibodies (Dako) were used at a 1:2,000 binding partner of EGFR (34, 35), OPCML restoration could dilution in either 5% BSA/TBST or 5% milk/TBST for incubation also prove effective for anti-EGFR therapy in HER2-positive for 1 hour at room temperature. Proteins were detected using cancer cells. Immobilon Western Chemiluminescent HRP Substrate system We therefore investigated this hypothesis in a panel of breast (Millipore) and developed on X-ray film using Kodak SRX2000 and ovarian cancer cell lines that do not express OPCML. Here we (Rochester) developer machine. Quantitative densitometry anal- show that OPCML restoration interferes with the HER2-EGFR ysis was carried out using ImageJ software (National Institute of heterodimer formation by interacting directly with HER2 but Health). not with EGFR, and that stably expressing OPCML in ovarian and breast cancer cells results in sensitization to HER2/EGFR Plasmid constructs (lapatinib) and EGFR-only (erlotinib) targeted therapies. Finally, The GST-OPCML construct was described previously (9). For we show that high endogenous OPCML expression is associated mammalian 2-hybrid studies, the domains 1, 2, and 3 of OPCML with better response to lapatinib in breast cancer patients and with were amplified by PCR from a human cDNA clone (pcDNA3.1- better progression free survival in ovarian cancer patients with OPCML) and sub-cloned into the pM vector using as forward high HER2 expression. primer 50-GggatccGTGCCACCTTCCCCAAAGCTATG-30 and as reverse primer 50-aagcttCCAGGCCCATACAATGTGATGC-30. The full ECD of p185neu/HER2 (NEX) was amplified by PCR from Materials and Methods the expression plasmid pNex (36) kindly provided by Prof. Cell lines and cell culture Mark Greene, University of Pennsylvania, using the forward SKOV3 cells (ATCC HTB-77) were maintained in RPMI medi- primer 50-gaattcCCCGGAATCGCGGGCACCC-30 and the reverse um (Sigma-Aldrich) supplemented with 10% FCS (First Link), primer 50-gtcgacCGTATACTTCCGGATCTTCTG-30. The full ECD 2 mmol/L L-glutamine (LG; Invitrogen), 50 U/mL Penicillin/ of EGFR was amplified by PCR from a human cDNA clone (EGFR- Streptomycin (Invitrogen). OV90 cells (ATCC CRL11732) were pcDNA3.1zeo, kindly provided by Prof. Bill Gullick, University of cultured in a 1:1 mixture of MCDB 105 medium (Sigma-Aldrich) Kent) using the forward 50-gtcgacGCCCGGCGAGTCGGGC-30 and Medium 199 (Sigma-Aldrich) supplemented with 15% and the reverse primer 50-aagcttGGACGGGATCTTAGGCCC-30. FCS, 2 mmol/L LG, 50 U/mL Penicillin/Streptomycin. SKBR3 The p185neu/HER2 and EGFR constructs were sub-cloned into the and MDA-MB-231 (ATCC HTB-26) cells were cultured in pVP16 vector. DMEM medium (Sigma-Aldrich) supplemented with 10% FCS, For virus production, p8.9 (gag/pol) and pmol/LDG (VSVG 2 mmol/L LG, 50 U/mL Penicillin/Streptomycin. Puromycin was envelope) plasmids were kindly provided by Dr. Ari Fassati,

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University College London, UK; the full-length OPCML sequence One Solution Cell Proliferation assay (Promega) following the was cloned into pLenti CMV Puro Dest that was purchased from manufacturer's recommendations. GI50 values were calculated Addgene. using the GraphPad Prism software. For Western blot analysis, cells were treated with targeted Pull-down assays therapy for 3 hours and then stimulated with 50 ng/mL rhEGF Assays were performed with recombinant GST-OPCML fusion (R&D systems) for 30 minutes before the cells were lysed for proteins bound to magnetic glutathione beads (Promega) as 20 minutes on ice. described previously (9). Lapatinib (Sigma-Aldrich) and erlotinib (VWR) were reconsti- tuted in dimethyl sulfoxide (DMSO; Sigma-Aldrich) and used at Mammalian 2-hybrid the concentrations shown. Each experiment was repeated three The three Ig domains of OPCML were cloned in the pM times and a representative figure from each experiment is included vector downstream of the SV40 promoter and GAL4 DNA– in the result section. binding domain, whereas the ECDs of EGFR and HER2 and the mutated HER2, lacking the ECD 4 (D4), were cloned in the Apoptosis assay pVP16 vector downstream of the SV40 promoter and VP16 Cells were seeded in triplicate in 96-well plates and treated with activation domain. COS-1 cells were transiently transfected at increasing concentrations of targeted therapy (0–120 mmol/L) for 80% confluence in 24-well plates for 2 to 3 hours with 50 ng of the 24 hours before assessing apoptosis by measuring caspase 3/7 pGAL4 Luc reporter, 50 ng of each of the different pM and pVP16 activation using the Caspase-Glo 3/7 assay (Promega) following constructs and 50 ng of the b-gal transfection control plasmid the manufacturer's instructions. Caspase activity was normalized using Effectene Transfection Reagent (Qiagen) at a 1:10 DNA to to cell density obtained by CellTiter 96 AQueous One Solution Cell Effectene reagent ratio. Cells were then washed with 1X PBS and Proliferation assay (Promega) for each treatment. Each experi- incubated in full medium overnight. Cells were collected in 1X ment was repeated three times. Cell Culture Lysis Reagent (CCLR) from the Promega Luciferase Assay System and incubated at 80C for 30 minutes. Luciferase Small interference RNA activity was measured by adding lysates and Luciferase Assay SKOV3 cells were transfected in 24-well plates with 20 nmol/L Substrate in Assay buffer (Promega) in equal volumes to white of siRNA 24 hours post trypsinization. After cells were harvested, flat-bottomed 96-well plates (PerkinElmer) followed by reading 2,000 cells per well were seeded in triplicate in 96-well plates X with a VICTOR Light Luminescence Plate Reader (PerkinElmer) for measuring cell viability by CellTiter 96 AQueous One Solution using the emission filter slot A7. Cell Proliferation assay (Promega) and 30,000 cells per well Transfection efficiency was measured using the Galacto-Light were seeded in 24-well plate for western blot analysis. siRNAs Plus beta-Galactosidase Reporter Gene Assay System (Applied containing a pool of siRNAs against EGFR2 (SMARTpool ON- Biosystems) according to the manufacturer's guidelines. TARGETplus L-003114) and HER2 (SMARTpool ON-TARGET- Each experiment was carried out in quadruplicate and the lucif- plus L-003126) were transfected either alone or in combination, erase measurements were corrected by dividing them for the non-targeting siRNA #2 was used as control. The siRNAs were b-galactosidase readings. Data presented are the mean of each transfected using Oligofectamine (Invitrogen, Life Technologies repetition normalized to the empty pM and pVP16 condition for Corporation) in Opti-MEM media (GIBCO, Thermofisher Scien- each experiment and are expressed as mean standard error of tific) according to the manufacturer's protocol. All siRNAs were mean (SEM). Student's unequal variances t tests (two-tailed) of purchased from Dharmacon (GE Healthcare Life Science). the data were performed to determine statistical significance of a positive protein–protein interaction using the OPCML-pM and Immunohistochemical analysis RTK-pVP16–negative controls as baseline. All statistical analysis Histopathological samples were obtained from 8 patients with was carried out in Microsoft Excel. advanced recurrent breast cancer that were treated previously with adjuvant chemotherapy and following treatment with lapatinib Duolink proximity ligation assay (Tyverb). The following information was extracted from the For proximity ligation assay (PLA assay; Duolink, OLink Bios- histopathology reports: tumor type, grade, stage, estrogen recep- ciences, Sigma-Aldrich), the various cell lines were seeded on tor (ER), progesterone receptor (PR), as well as HER2 immuno- 13 mm glass coverslips. After the required treatments, cells were scores. Sections of 2-mm thickness from each tumor were cut from fixed with ice-cold methanol for 5 minutes at 20 C, washed with paraffin wax-embedded blocks, floated onto polysine slides, and PBS and fixed with 3% BSA in PBS for 30 minutes at room dried overnight at 37C. The next day, sections were de-waxed, temperature. Cells were then labeled with primary antibodies rehydrated, and incubated with 0.6% peroxide block for 15 min- diluted 1:100 in 1% BSA/PBS at room temperature for 1 hour, utes to block endogenous peroxidase activity. Antigen retrieval taking care to use two different species (e.g., Mouse anti-HER2 and was performed by microwaving the samples for 20 minutes in Rabbit anti-EGFR). Incubation with PLA probes MINUS and EDTA (pH 9.0) at 1,000 W. Background staining was blocked PLUS, ligation and amplification steps were carried out as per using Power block (protein block supplied by Biogenex) for the manufacturer's instructions. Quantification of the number of 10 minutes. The sections were then incubated with the mono- Duolink particles was performed using the ImageJ software. clonal anti-human OPCML antibody (R&D Systems) at 1 mg/mL for 1 hour at room temperature and detected using Super- Drug treatment and proliferation assay Sensitive Polymer-HRP detection kit with diaminobenzidine Cells were seeded in triplicate in 96-well plates and treated with substrate at the manufacturer's instructions (Biogenex, Launch increasing concentrations of targeted therapy (0–100 mmol/L) for Diagnostics). Counterstaining was carried out with hematoxylin. 48 hours before measuring cell viability by CellTiter 96 AQueous For negative controls, duplicate sections were used in which the

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OPCML Sensitizes HER2þ Cancers to Anti-HER2/EGFR Therapy

primary antibody was omitted and replaced with PBS. Sections OPCML potentiates lapatinib and erlotinib therapy in ovarian were examined by light microscopy and staining was assessed and breast cancer cell lines overexpressing HER2 with respect to presence, localization and intensity of staining. As we observed previously, OPCML binding to HER2 down- Staining was scored as weak (þ), moderate (þþ) or strong regulates the receptor and its downstream signaling in ovarian (þþþ). Ethical approval to carry out expression analysis on cancer cell lines (9). This led us to examine the potential thera- snap-frozen samples from breast cancer patients was approved peutic benefits of OPCML when combined with anti-HER2 by Hammersmith and Queen Charlotte's and Chelsea Research agents. We first evaluated the impact of OPCML expression on Ethics Committee (REC reference 05/Q0406/178). the effect of the dual anti-HER2/EGFR small-molecule TKI lapatinib. Two ovarian cancer cell lines (SKOV3 and OV90) and a breast cancer cell line (SKBR3) with varying levels of EGFR and Results HER2 expression and with low or no HER3 and HER4 expression OPCML binds to HER2 but not EGFR, interfering with the (Supplementary Fig. S2) were stably transduced to express formation of the HER2-EGFR heterodimer OPCML (Supplementary Fig. S3) and treated with increasing Our previous co-immunoprecipitation (co-IP) experiments amounts (0–100 mmol/L) of lapatinib for 48 hours before mea- suggested that OPCML interacts with HER2, but not with suring their cell viability. In cells overexpressing HER2 (SKOV3 EGFR, in the ovarian cancer cell line SKOV3 (9). Here, we have and SKBR3), we observed a significant decrease in cell viability investigated more in depth this difference by using a recombinant when OPCML was present in the cells (control GI50 ¼ 34 mmol/L GST-OPCML to perform pull-down experiments in two breast vs. OPCML GI50 ¼ 15.5 mmol/L for SKOV3, and control GI50 ¼ cancer cell lines with no detectable OPCML expression: SKBR3, 13.6 mmol/L vs. OPCML GI50 ¼ 1 mmol/L in SKBR3), while in cells which overexpresses HER2 and is positive for EGFR, and MDA- with little HER2 expression (OV90) the significance was lost even MB-231, positive for EGFR and expressing very low levels of though a similar trend was maintained (Fig. 2A). We then ana- HER2. In SKBR3 cells, we found that HER2 was efficiently lyzed the downstream signaling of these cell lines treated with pulled-down in the cell lysate by GST-OPCML (Fig. 1A), but no serial dilutions of lapatinib for 3 hours followed by stimulation EGFR could be detected (Fig. 1B), thus validating our previous with EGF for 30 minutes. Lapatinib effects on the tyrosine phos- findings. The pull-down assay was then repeated in the HER2- phorylation levels of HER2 were very obvious at the lowest negative MDA-MB-231 breast cancer cell line and again no EGFR concentration of the drug (25 nmol/L) whereas phospho-EGFR could be detected (Fig. 1B), thus providing further evidence that levels were mostly inhibited at the highest concentrations (100– OPCML does not interact with EGFR. 200 nmol/L; Fig. 2C and D), with the total levels of proteins being To further confirm these results, we carried out mammalian unaffected (Supplementary Fig. S4). These results confirm previ- 2-hybrid studies. The ECDs of HER2 and EGFR were cloned ous findings, suggesting that lapatinib mainly acts through HER2 to determine their ability to interact with OPCML (Supplemen- rather than EGFR (38). Overall, OPCML-expressing cells had tary Fig. S1) and transiently transfected in Cos-1 cells, which reduced amounts of the survival downstream effector phos- have a low basal expression of both HER2 and EGFR. The pho-AKT at lower concentrations of the drug compared with luminescence was normalized by co-transfection with a plas- control cells in SKOV3 and SKBR3 and, at a lesser level, in OV90 b mid expressing the -galactosidase gene and the empty pM and cells (Fig. 2C and D), correlating with the GI50 data. The effects of pVP16 vectors were used as negative controls. As presented lapatinib on the mitogen-activated protein kinase (MAPK) path- in Fig. 1C, a positive signal was obtained only when cells were way were also analyzed but only OPCML-expressing SKOV3 co-transfecte with OPCML and HER2,confirming again that cells showed reduction of the phospho-ERK1/2 signal compared OPCML interacts with HER2, but not with EGFR. Furthermore, with control cells (Fig. 2C and D). The consistent downregulation we investigated which portion of the receptor was important for of the phospho-AKT signal at lower doses of lapatinib mediated the binding to OPCML and deletion of domain 4 of the ECD of by OPCML in SKOV3 and SKBR3 cells was also reflected HER2 (HER2 D4, Supplementary Fig. S1) was shown to disrupt in activation of the apoptotic pathway in both cell lines. Caspase theinteraction(Fig.1D).Asthemembrane-proximal portion of 3 and 7 activation after 24 hours of treatment with lapatinib, domain 4 of EGFR and HER2 has been proposed to mediate was in fact strongly increased and detected at lower concentrations part of the interaction between the two molecules (37), we then of the drug in the OPCML-expressing cells compared with control analyzed the ability of the two receptors to heterodimerize in (Fig. 2B). These results suggest that OPCML sensitizes cells the presence or absence of OPCML. In SKOV3 and SKBR3 cells, to lapatinib mainly through pAKT inhibition- and caspase an ovarian and a breast cancer cell line, respectively, that activation- pathway. express high levels of both HER2 and EGFR, HER2 was co- Because OPCML binds to HER2 making it less available for immunoprecipitated by an anti-EGFR antibody three times less hetero-dimerization with EGFR (Fig. 1), a therapy that targets only in the presence of OPCML expression compared with OPCML EGFR could still benefit from the presence of OPCML, even non-expressing control cells (Fig. 1E). We then confirmed these though OPCML does not affect EGFR levels directly. We investi- results further and quantified the interaction by using a prox- gated our hypothesis by treating the cells with the anti-EGFR imity ligation assay (PLA). The Duolink signal given by the kinase inhibitor erlotinib for growth inhibition studies and found close vicinity of EGFR to HER2 in non-stimulated cells was that, similarly to what seen for lapatinib, SKOV3 and SKBR3 cells maintained in control cells stimulated with EGF for 30 minutes expressing OPCML had a significant decrease in cell viability and dropped after one-hour stimulation, while in OPCML- compared with control cells, while in OV90 cells this difference expressing cells it was rapidly and strongly reduced by 30 min- was not apparent (Fig. 3A). However, in OPCML-expressing utes (Fig. 1F). This suggests that OPCML interaction with HER2 cells the degree of sensitization to erlotinib (Control GI50 ¼ induces a reduction of the HER2/EGFR dimer through dimer 32.5 mmol/L and 24.3 mmol/L vs. OPCML GI50 ¼ 24.5 mmol/L formation interference. and 19.4 mmol/L for SKOV3 and SKBR3, respectively) was

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Figure 1. OPCML binds to HER2 but not to EGFR and interferes with the HER2/EGFR dimer formation. HER2 (A) and EGFR (B) were pulled down by GST-OPCML from two breast cancer cell lines, MDA-MB-231 and SKBR3, and analyzed by Western blot. GST only was used as a control. C, The interaction between OPCML and the ECD of HER2 and EGFR was detected by mammalian 2-hybrid. Luciferase activity was normalized to b-galactosidase activity to give the relative activity. Results shown are the mean of three independent experiments performed in quadruplicates SEM; , P < 0.05; , P < 0.0005. D, The interaction between OPCML and HER2 lacking domain 4 of the ECD (~4) was detected by mammalian 2-hybrid. Luciferase activity was normalized to b-galactosidase activity to give the relative activity. Results shown are the mean of three independent experiments performed in quadruplicates SEM; , P < 0.05; , P < 0.005. E, Co-IP experiments in ovarian cancer cell line SKOV3 and breast cancer cell line SKBR3 stably expressing OPCML or control vector (CTR). HER2 and EGFR interaction was quantiﬁed by dividing the intensity of the prey protein by the intensity of the relative bait protein. IP: Immunoprecipitated, IB: immunoblotted. F, Duolink experiments in SKOV3 cells stably expressing OPCML or control vector (CTR) stimulated with 50 ng/mL EGF for 0, 30 minutes, or 1 hour. Duolink particles were quantiﬁed using the ImageJ software.

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OPCML Sensitizes HER2þ Cancers to Anti-HER2/EGFR Therapy

Figure 2. OPCML sensitizes HER2-positive ovarian and breast cancer cells to lapatinib through caspase 3/7 activation. A, SKOV3, OV90, and SKBR3 cells (control or stably expressing OPCML) were treated with serial dilutions (0–100 mmol/L) of lapatinib for 48 hours; cell viability was measured by MTS and used to calculate GI50 values. Three independent experiments were performed, and significant differences were determined by t-test analysis; , P < 0.005; , P < 0.0005. B, Caspase 3/7 assays in SKOV3 and SKBR3 cells (control or stably expressing OPCML) treated with serial dilutions (0–70 mmol/L for SKOV3 and 0–120 mmol/L for SKBR3) of lapatinib for 24 hours. Caspase activity was normalized to cell density obtained by MTS for each treatment. Three independent experiments were performed, and significant differences were determined by t test analysis; , P < 0.05; P < 0.005; , P < 0.0005. C, SKOV3, OV90, and SKBR3 cells were also treated with serial dilutions of lapatinib for 3 hours and then stimulated with 50 ng/mL of rhEGF for 30 minutes, and the downstream signaling pathway was analyzed by Western blot. D, pHER2, pEGFR, pAKT, and pERK1/2 signals obtained by densitometry analysis were normalized to calnexin and to untreated CTR cells (þ/D). D: DMSO. much less compared with the degree of sensitization to lapa- lower doses of erlotinib in OPCML-expressing SKOV3 and tinib, highlighting that OPCML confers a more efficient sensi- SKBR3 but not in OV90 cells (Fig. 3C and D). Here, tization of anti-HER2 therapy rather than anti-EGFR. Western OPCML-mediated reduction of the phospho-ERK1/2 signal was blot analysis of the downstream signaling of these cell lines found not only in SKOV3 cells but also, at a lower extent, in treated with erlotinib for 3 hours and stimulated with EGF for SKBR3 cells (Fig. 3C and D). The ability of the SKBR3 cells to 30 minutes confirmed the cell viability results, in which the respond to EGFR-targeted therapy despite lack of downregula- phospho-HER2 and phospho-AKT levels were inhibited at tion of the phospho-EGFR levels (Fig. 3D) has also been found

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Figure 3. OPCML sensitizes HER2-positive ovarian and breast cancer cells to erlotinib through caspase 3/7 activation. A, SKOV3, OV90, and SKBR3 cells (control

or stably expressing OPCML) were treated with serial dilutions (0–100 mmol/L) of erlotinib for 48 hours, cell viability was measured by MTS and used to calculate GI50 values. Three independent experiments were performed, and signiﬁcant differences were determined by t test analysis; , P < 0.05; , P < 0.005. B, Caspase 3/7 assays in SKOV3 and SKBR3 cells (control or stably expressing OPCML) treated with serial dilutions (0–96 mmol/L) of erlotinib for 24 hours. Caspase activity was normalized to cell density obtained by MTS for each treatment. Three independent experiments were performed, and signiﬁcant differences were determined by t test analysis; , P < 0.05. C, SKOV3, OV90, and SKBR3 cells were also treated with serial dilutions of erlotinib for 3 hours and then stimulated with 50 ng/mL of rhEGF for 30 minutes, and the downstream signaling pathway was analyzed by Western blot. D, pHER2, pEGFR, pAKT, and pERK1/2 signals obtained by densitometry analysis were normalized to calnexin and to untreated CTR cells (þ/D). D: DMSO.

by others (39) and has been suggested as an evidence for These observations were further confirmed by genetic inhi- sensitivity of HER2-overexpressing tumors to EGFR-specific bition through siRNA of HER2, EGFR or both. When either TKIs (39–41). Caspase 3/7 activation assays in SKOV3 and HER2 or EGFR or both were silenced (Fig. 4A), the viability of SKBR3 cells treated with erlotinib for 24 hours confirmed the SKOV3 cells decreased significantly compared with a non- GI50 data and OPCML-expressing cells showed higher levels of targeting control (siNT) after 48 and 72 hours from transfection the activated enzymes compared with control cells (Fig. 3B) (Fig. 4B). However, OPCML-expressing cells showed a further However, this was to a much lower extent compared with decrease in cell viability at 72 hours compared with control lapatinib treatment, suggesting again that the combined cells when HER2, but not EGFR, protein levels were down- HER2-targeted therapy is more efficient in killing cancer cells. regulated (Fig. 4B).

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OPCML Sensitizes HER2þ Cancers to Anti-HER2/EGFR Therapy

Figure 4. OPCML further inhibits cell growth after knockdown of HER2. A, Western blot analysis of the knockdown of EGFR, HER2 or both in SKOV3 cells. NT: non-targeting. B, After knockdown of EGFR, HER2 or both (siEGþsiH, E), cell viability was measured at 48 and 72 hours in SKOV3 control (CTR) and OPCML cells. Three independent experiments were performed and signiﬁcant differences were determined by t test analysis; , P < 0.05.

Higher expression levels of OPCML correlate with better the interference with the EGFR/HER2 heterodimer formation is response to lapatinib in breast cancer patients and better mediated by the binding of OPCML to the domain 4 of the ECD of survival in HER2-positive ovarian cancer patients HER2, which is important for the stabilization of the heterodimer In view of the above findings, we evaluated the predictive value (37). Given these results, we have then investigated the ability of þ of OPCML protein expression in a series of known HER2 OPCML to sensitize cells to HER2- and EGFR-targeted therapies in recurrent breast cancer patients receiving capecitabine and lapa- HER2-overexpressing cancer cell models. By acting on HER2, tinib. We had previously demonstrated that OPCML mRNA OPCML affects the signaling from the EGF receptor family, as expression is a significant good prognostic factor for recurrence HER2 is the preferential partner of the three other members of the free survival in a series of over 2,300 breast cancer patients (9). family (34, 35), as it is always in an open conformation (43, 44). þ HER2 (all cases 3þ) breast cancer patients treated with capeci- We therefore used lapatinib (EGFR/HER2) and erlotinib (EGFR- tabine and lapatinib were selected (Supplementary Fig. S5). only) RTK inhibitors in cancer cells bearing varying levels of Following immunohistochemistry (IHC) staining for OPCML expression of these two receptors (and no or little expression of (Supplementary Fig. S6), this small series demonstrated a statis- HER3/4) in which we then re-expressed OPCML. Lapatinib acts as tically significant correlation between OPCML protein expression an ATP competitor for the kinase domain of both HER2 and EGFR and RECIST response to lapatinib/capecitabine (Fisher's Exact causing the downregulation of the mitogenic and proliferative Test, two tailed P ¼ 0.0286). All patients with 2þ and 3þ OPCML IHC expression responded, whereas none of the patients with 0 PFS_OvCa_OPCML_HER2 and 1þ OPCML IHC expression did (Supplementary Fig. S5). We then looked at progression-free survival (PFS) of ovarian 1.0 Low Her2/Low OPCML cancer patients with HER2 overexpression (top quartile) in the Low Her2/High OPCML fi High Her2/Low OPCML Tothill dataset (42) and found that patients had a signi cantly High Her2/High OPCML better survival when OPCML expression was above median (log- 0.8 rank P ¼ 0.004). In patients with low HER2 (bottom quartile) OPCML expression did not improve survival (Fig. 5). fi P Overall these data further con rm OPCML relevance in the 0.6 Log-rank = 0.004 regulation of HER2 signaling in a clinical setting.

Discussion 0.4 OPCML is a protein that is frequently lost in tumors due to a combination of LOH and hypermethylation of the promoter of Progression-free survival 0.2 the gene (1, 2). We previously described how its tumor-suppres- sing function is achieved, showing that, by interacting with and downregulating a speciﬁc repertoire of RTKs, it causes a profound 0.0 inhibition of the downstream ERK and AKT pathways (9). In 0 10 20 30 40 50 60 particular, OPCML downregulates HER2 by changing the receptor Time (months) localization on the cell membrane and inducing its internaliza- – tion through a non clathrin-mediated pathway (9). Here, we Figure 5. ﬁ have con rmed by pull-down and mammalian 2-hybrid assays High expression of OPCML in HER2-positive ovarian cancers is associated the interaction between OPCML and HER2 and demonstrated by with better progression-free survival. Kaplan–Meier survival curves co-IP experiments and Duolink assays that OPCML prevents the of 254 ovarian cancer patients from Tothill's dataset based on OPCML (median) heterodimerization of HER2 with EGFR. We have also shown that and HER2 (top and bottom quartiles) expression.

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Figure 6. Schematic diagram of OPCML as a sensitizing agent. A, Ligand binding to the ECD of EGFR triggers the formation of heterodimers with HER2 on the cell membrane, causing the activation of downstream signals. B, Erlotinib and lapatinib are small-molecule inhibitors that target the cytoplasmic domain of EGFR only or both EGFR and HER2, respectively, interfering with receptor trans-activation and causing the downregulation of intracellular signaling. C, OPCML, by binding to HER2, interferes with the receptors heterodimerization increasing the effects of both erlotinib and lapatinib on cell survival.

signaling driven by the activation of the two receptors (45). inhibitor. The analysis of survival pathways mediated by AKT Despite the promising initial response of patients to this drug, confirmed that cells expressing OPCML had a significant reduc- relapse and resistance are still common events. It has been then tion in phospho-AKT levels at lower doses of lapatinib compared suggested that the combination of HER2-targeting therapies can with control cells in both SKOV3 and SKBR3 cells and to a much be more effective in the initial line of treatment as well as in lesser extent in OV90 cells, which have low expression levels of relapsing cancers (18). In this study, we have shown how re- HER2. When treating the same cell lines with the EGFR inhibitor expression of OPCML in ovarian and breast cancer cell lines with erlotinib we obtained similar results, with cells overexpressing high HER2 levels (SKOV3 and SKBR3) sensitizes these cells to HER2 being sensitized to the drug through a higher activation of lapatinib through caspase 3/7 activation. This confirms that by caspase 3/7 and the phospho-AKT signaling being strongly affect- interfering with both HER2 dimerization (through OPCML inter- ed by OPCML expression in SKOV3 and SKBR3 cells and only action with the ECD) and kinase activation (through lapatinib partially in OV90 cells. However, overall the magnitude of sen- action) cells die at significantly lower concentrations of the sitization to erlotinib was lower compared with lapatinib,

2254 Mol Cancer Ther; 16(10) October 2017 Molecular Cancer Therapeutics

OPCML Sensitizes HER2þ Cancers to Anti-HER2/EGFR Therapy

suggesting that OPCML targeting of HER2 is indeed a more Disclosure of Potential Conflicts of Interest efficient approach to inhibit cancer cell growth. H. Gabra is a VP head of Oncology Clinical Discovery (concurrent position These findings were also corroborated in a clinical setting, with an Imperial tenured chair in medical oncology) at AstraZeneca and has where breast cancer patients with HER2-positive tumors were ownership interest (including patents) in Patent to develop OPCML-based therapeutics. No potential conflicts of interest were disclosedby the other authors. found to be significantly more likely to respond to capecitabine/ lapatinib therapy in the context of high tumoral OPCML protein Authors' Contributions expression levels compared with OPCML non/weakly expressing Conception and design: E. Zanini, L.S. Louis, E. Karali, I.S. Okon, A.B. McKie, patients. Furthermore, stratification for HER2 and OPCML expres- S. Vaughan, J. Stebbing, C. Recchi, H. Gabra sion of ovarian cancer patients showed that higher co-expression Development of methodology: L.S. Louis, E. Karali, I.S. Okon, J. Stebbing, of OPCML significantly increased progression-free survival of H. Gabra HER2-overexpressing patients. Acquisition of data (provided animals, acquired and managed patients, Taken together these results show how, by acting through a key provided facilities, etc.): E. Zanini, L.S. Louis, J. Antony, M. El-Bahrawy, molecule such as HER2, OPCML amplifies the anticancer effect of H. Gabra Analysis and interpretation of data (e.g., statistical analysis, biostatistics, lapatinib and to a lesser extent erlotinib and allows for a more computational analysis): E.Zanini,L.S.Louis,J.Antony,E.Karali,M.El-Bahrawy, effective targeting strategy of the EGFR-HER2 axis (Fig. 6). H. Gabra Furthermore, these results support the notion of OPCML as a Writing, review, and/or revision of the manuscript: E. Zanini, L.S. Louis, sensitizing agent in cancers with an activated HER2 status. There- A.B. McKie, M. El-Bahrawy, J. Stebbing, C. Recchi, H. Gabra fore, we hypothesize that selection by OPCML expression status Administrative, technical, or material support (i.e., reporting or organizing þ might enrich for RTK inhibitor responders in HER2 breast data, constructing databases): E. Zanini, J. Antony, H. Gabra Study supervision: E. Zanini, L.S. Louis, S. Vaughan, C. Recchi, H. Gabra and ovarian cancer patients, although this requires further vali- Other (Joint Fisrt Author with E Zanini): L.S. Louis dation in the future. In addition, we previously demonstrated that a recombinant OPCML biotherapeutic was an active anti- Acknowledgments in-vivo cancer agent preclinically (9) and therefore this biother- We wish to acknowledge the support of the NIHR Biomedical Research apeutic could be used in combination with HER2 targeting Centre, the Cancer Research UK Clinical Centre, and the Experimental Cancer TKIs to enhance their response. Considering that OPCML also Medicine Centre at Imperial College London. downregulates other RTKs, these data also open up the possibility that this general effect of OPCML may apply to other RTK targeting Grant Support scenarios, where different RTKs might be driving cancer growth. E. Zanini, L.S. Louis, J. Antony, E. Karali, I.S. Okon, A.B. McKie, S. Vaughan, By acting at several levels in the RTK crosstalk, OPCML could and C. Recchi were supported by the Ovarian Cancer Action Research Centre delay or even prevent the onset of resistance that often follows the Core Funding (principal investigator: H. Gabra). The costs of publication of this article were defrayed in part by the payment of use of RTK inhibitors due to compensatory mechanisms. OPCML page charges. This article must therefore be hereby marked advertisement in could therefore prove valuable as a therapeutic not just in RTK accordance with 18 U.S.C. Section 1734 solely to indicate this fact. addicted cancers but also in cancers that are not strictly addicted to a specific oncogene, allowing responses that would not otherwise Received January 30, 2017; revised May 22, 2017; accepted July 14, 2017; occur. published OnlineFirst August 3, 2017.

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2256 Mol Cancer Ther; 16(10) October 2017 Molecular Cancer Therapeutics

The Tumor-Suppressor Protein OPCML Potentiates Anti−EGFR- and Anti −HER2-Targeted Therapy in HER2-Positive Ovarian and Breast Cancer

Elisa Zanini, Louay S. Louis, Jane Antony, et al.

Mol Cancer Ther 2017;16:2246-2256. Published OnlineFirst August 3, 2017.

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