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

Author Manuscript Published OnlineFirst on August 3, 2017; DOI: 10.1158/1535-7163.MCT-17-0081 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

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 Stebbing4, Chiara Recchi1§ and Hani Gabra1, 5§

1Ovarian Cancer Action Research Centre, Department of Surgery and Cancer, Imperial College London, London, UK 2Center for Molecular and Translational Medicine, Georgia State University, Atlanta, USA 3Department of Medical Genetics, University of Cambridge, Addenbrooke’s Treatment Centre, Cambridge Biomedical Campus, Cambridge, UK 4Department of Histopathology, Imperial College London, London, UK 5Clinical Discovery Unit, Early Clinical Development, AstraZeneca, Cambridge

* contributed equally

§ corresponding authors

Running title: OPCML sensitizes HER2+ cancers to anti-HER2/EGFR therapy.

Keywords: OPCML, HER2, EGFR, lapatinib, erlotinib.

Grant Support: EZ, LSL, JA, EK, ISO, ABM, SB, CR were supported by the Ovarian Cancer Action Research Centre Core Funding (principal investigator H. Gabra).

Corresponding authors: Chiara Recchi and Hani Gabra, Ovarian Cancer Action Research Centre, Department of Surgery and Cancer, Imperial College London, Du Cane Road, London W12 0NN, United Kingdom. Phone: 44–208– 383–5828; Fax: 44–208–383–5830; E-mail: [email protected], [email protected]

Word count: 4669

Total number of figures: 6 (+6 supplementary)

Downloaded from mct.aacrjournals.org on September 27, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 3, 2017; DOI: 10.1158/1535-7163.MCT-17-0081 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract OPCML is a tumor suppressor gene that is frequently inactivated in ovarian cancer and many other cancers by somatic methylation. We have previously shown that OPCML exerts its suppressor function by negatively regulating a spectrum of receptor tyrosine kinases (RTKs), such as ErbB2/HER2, FGFR1 and EphA2, thus attenuating their related downstream signaling. The physical interaction of OPCML with this defined group of RTKs is a prerequisite for their downregulation. Overexpression/gene amplification of EGFR and HER2 is a frequent event in multiple cancers including ovarian and breast cancers. Molecular therapeutics against EGFR/HER2 or EGFR only, such as lapatinib and erlotinib respectively, were developed to target these receptors but resistance often occurs in relapsing cancers. Here we show that, though OPCML interacts only with HER2 and not with EGFR, the interaction of OPCML with HER2 disrupts the formation of the HER2-EGFR heterodimer and this translates into a better response to both lapatinib and erlotinib in HER2-expressing ovarian and breast cancer cell lines. Also, we show that high OPCML expression is associated with better response to lapatinib therapy in breast cancer patients and better survival in HER2-overexpressing ovarian cancer patients, suggesting that OPCML co-therapy could be a valuable sensitizing approach to RTK inhibitors. (Word count: 200)

Introduction Opioid-binding protein/cell adhesion molecule-like (OPCML) is a glycosyl phosphatidylinositol (GPI) anchored molecule that is expressed in virtually all normal adult and foetal tissues (1). We first identified OPCML as a putative tumor suppressor gene (TSG) at chromosome 11q25, a site highlighted by frequent loss of heterozygosity (LOH), and further demonstrated that epigenetic silencing of the remaining OPCML allele occurred in 83% of epithelial ovarian cancers (EOC)(2). OPCML methylation in EOC has also been confirmed by other studies (3,4). Furthermore, OPCML is inactivated in numerous other types of cancers, such as brain, nasopharyngeal, oesophageal, gastric, hepatocellular, colorectal, breast and cervical cancers, as well as lymphomas (1,4–6). In lung adenocarcinoma, OPCML is one of the four most commonly methylated genes compared with normal lung tissue (7,8). All these data indicate that OPCML expression is 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 (RTKs), including HER2, HER4, EphA2, FGFR1 and FGFR3 (9). In particular, OPCML binding to the extracellular domain of HER2 promotes HER2 ubiquitination and proteasomal degradation via clathrin independent

endocytosis (9). Conversely, silencing of OPCML causes up-regulation of this same repertoire of RTKs and increased signaling (9). OPCML therefore induces a system level switch-off of a spectrum of RTKs, which eventually leads to a strong decrease in phosphorylation and activation of key downstream signaling molecules such as ERK and AKT, thus reducing cell proliferation in vitro and in vivo (9). HER2 is an RTK whose overexpression drives cell proliferation in many human cancers. Studies have shown that ovarian tumors with high malignant potential over-express HER2 in 22 – 66% of cases (10), although recent consensus reduces the number of cases with genomic amplification of HER2 to 11% (11). In breast cancer, HER2 amplification is reported in 15-20% of cases (12). In comparison to the above, EGFR over-expression can be as high as 28% and amplified in up to 20% of ovarian cancer patients (13) and 13 – 52% in triple negative breast cancer (14). The concept of “oncogene addiction”, by which the growth of some tumors relies heavily on the amplification of a single oncogene (15), as well as its downstream pathway, led to the development of targeted therapies directed against HER2 and EGFR. Trastuzumab (a monoclonal antibody targeting HER2) and lapatinib (a small molecule inhibitor against HER2 and EGFR) have been used successfully in the treatment of metastatic HER2 positive breast cancer along with chemotherapy, resulting in increased response rate, prolonged progression free survival and extended overall survival compared with chemotherapy alone (16,17). However, only 20 – 30% of HER2 positive breast tumors respond to single HER2-targeted therapy (18) and those that initially respond eventually develop recurrence due to drug resistance (19). Erlotinib (Tarceva, an EGFR- specific inhibitor) has been approved for the treatment of recurrent Non-Small Cell Lung Cancer (NSCLC)(20) with a specific EGFR mutation (21,22). Despite the positive initial response, also these patients develop resistance after 10-13 months of treatment with erlotinib (23). Mechanisms of resistance to tyrosine kinase inhibitors (TKIs) include the activation of alternative pathways through RTK cross-talk (24–29) and mutations in the tyrosine kinase domain of the receptors (30–32). Several alternative approaches have been proposed to overcome drug resistance, such as the concomitant use of lapatinib and

trastuzumab upfront and the creation of new small molecule inhibitors (33), and these are currently being evaluated. Since we have shown that the expression of OPCML is a good prognostic factor for progression free survival in ovarian and breast cancer patients and that it induces HER2 down-regulation in ovarian cancer cells (9), OPCML restoration could represent a novel approach to reduce the level of activity of this oncogene and thereby sensitize cancer cells to anti-HER2 therapy. Furthermore, as HER2 is the preferred binding partner of EGFR (34,35), OPCML restoration could also prove effective for anti-EGFR therapy in HER2-positive cancer cells. We therefore investigated this hypothesis in a panel of breast and ovarian cancer cell lines that do not express OPCML. Here we show that OPCML restoration interferes with the HER2-EGFR heterodimer formation by interacting directly with HER2 but not with EGFR, and that stably expressing OPCML in ovarian and breast cancer cells results in sensitization to HER2/EGFR (lapatinib) and EGFR- only (erlotinib) targeted therapies. Finally, we show that high endogenous OPCML expression is associated with better response to lapatinib in breast cancer patients and with better progression free survival in ovarian cancer patients with high HER2 expression.

Materials and Methods Cell lines and cell culture SKOV3 cells (ATCC® HTB-77TM) were maintained in RPMI medium (Sigma- Aldrich) supplemented with 10% Fetal Calf Serum (FCS) (First Link), 2 mM L- glutamine (LG) (Invitrogen), 50 U/ml Penicillin/Streptomycin (Invitrogen). OV90 cells (ATCC® CRL11732TM) were cultured in a 1:1 mixture of MCDB 105 medium (Sigma-Aldrich) and Medium 199 (Sigma-Aldrich) supplemented with 15% FCS, 2 mM LG, 50 U/ml Penicillin/Streptomycin. SKBR3 and MDA-MB-231 (ATCC® HTB-26TM) cells were cultured in DMEM medium (Sigma-Aldrich) supplemented with 10% FCS, 2 mM LG, 50 U/ml Penicillin/Streptomycin. Puromycin was used at 1μg/ml as a selective antibiotic for SKOV3 cell lines, at 0.1μg/ml for OV90 cells and at 0.2 μg/ml for SKBR3. COS-1 simian kidney fibroblast cells (CV-1) were maintained in DMEM, supplemented with 10% FCS, 2 mM LG, 50 U/ml Penicillin/Streptomycin.

All cells were tested regularly to exclude mycoplasma infection. COS-1 cells were tested and authenticated as described previously (9). All the other cell lines were purchased from the American Type Culture Collection (ATCC) in January 2013.

Antibodies Polyclonal goat Anti Human OBCAM (OPCML) antibody was purchased from R&D Systems. Total and phospho-HER2 (Y1248), total and phospho-EGFR (Y1068), total and phospho-AKT (S473) antibodies were purchased from Cell Signaling. Total HER2 antibody (OP16L) used for Duolink® was purchased from Merck Millipore. Calnexin antibody was purchased from ENZO Life Sciences.

Western blotting Prior to collection, cells were washed in phosphate buffered saline (PBS). Whole cell lysates were collected in RIPA buffer (Sigma-Aldrich) supplemented with protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail II (Calbiochem) at the manufacturers’ recommended concentration. Protein concentration was estimated using the BCA assay (Pierce) according to the manufacturer’s recommendations. Lysates were incubated at 950C for 5 min and then separated into 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Gels were then transferred onto nitrocellulose membranes (VWR) that were then blocked with 5% milk/TBST. Membranes were incubated with primary antibodies in 5% BSA/TBST or 5% milk/TBST at 4°C overnight. HRP-conjugated secondary antibodies (Dako) were used at a 1:2000 dilution in either 5% BSA/TBST or 5% milk/TBST for incubation for 1 hour at room temperature. Proteins were detected using ImmobilonTM Western Chemiluminescent HRP Substrate system (Millipore) and developed on X-ray film using Kodak SRX2000 (Rochester, NY, USA) developer machine. Quantitative densitometry analysis was carried out using ImageJ software (National Institute of Health, Bethesda, MD, USA).

Plasmid constructs The GST-OPCML construct was described previously (9).

For mammalian 2-hybrid studies, the domains 1, 2 and 3 of OPCML were amplified by PCR from a human cDNA clone (pcDNA3.1-OPCML) and sub-cloned into the pM vector using as forward primer 5’- GggatccGTGCCACCTTCCCCAAAGCTATG-3’ and as reverse primer 5’- aagcttCCAGGCCCATACAATGTGATGC-3’. The full extracellular domain of p185neu/HER2 (NEX) was amplified by PCR from the expression plasmid pNex (36) kindly provided by Prof. Mark Greene, University of Pennsylvania, using the forward primer 5’-gaattcCCCGGAATCGCGGGCACCC-3’ and the reverse primer 5’- gtcgacCGTATACTTCCGGATCTTCTG-3’. The full extracellular domain of EGFR was amplified by PCR from a human cDNA clone (EGFR-pcDNA3.1zeo, kindly provided by Prof. Bill Gullick, University of Kent) using the forward 5’- gtcgacGCCCGGCGAGTCGGGC-3’ and the reverse primer 5’- aagcttGGACGGGATCTTAGGCCC-3’. The p185neu/HER2 and EGFR constructs were sub-cloned into the pVP16 vector. For virus production, p8.9 (gag/pol) and pMDG (VSVG envelope) plasmids were kindly provided by Dr. Ari Fassati, University College London, UK; the full length OPCML sequence was cloned into pLenti CMV Puro Dest that was purchased from Addgene.

Pull-down assays Assays were performed with recombinant GST-OPCML fusion proteins bound to magnetic glutathione beads (Promega) as described previously (9).

Mammalian 2-hybrid The three Ig domains of OPCML were cloned in the pM vector downstream of the SV40 promoter and GAL4 DNA binding domain, while the extracellular domains of EGFR and HER2 and the mutated HER2, lacking the extracellular domain 4 ('4), were cloned in the pVP16 vector downstream of the SV40 promoter and VP16 activation domain. COS-1 cells were transiently transfected at 80% confluence in 24 well-plates for 2-3 h with 50 ng of the pGAL4 Luc reporter, 50 ng of each of the different pM and pVP16 constructs and 50 ng of the β-gal transfection control plasmid using Effectene® Transfection Reagent (Qiagen) at a 1:10 DNA to Effectene reagent ratio. Cells were then washed with 1X PBS and

incubated in full medium over-night. Cells were collected in 1X Cell Culture Lysis Reagent (CCLR) from the Promega Luciferase Assay System and incubated at - 80˚C for 30 min. Luciferase activity was measured by adding lysates and Luciferase Assay Substrate in Assay buffer (Promega) in equal volumes to white flat-bottomed 96 well-plates (PerkinElmer) followed by reading with a VICTOR™ XLight Luminescence Plate Reader (PerkinElmer) using the emission filter slot A7. Transfection efficiency was measured using the Galacto-Light Plus™ beta- Galactosidase Reporter Gene Assay System (Applied Biosystems) according to the manufacturer’s guidelines. Each experiment was carried out in quadruplicate and the luciferase measurements were corrected by dividing them for the β- galactosidase readings. Data presented are the mean of each repetition normalised to the empty pM and pVP16 condition for each experiment and are expressed as mean ± standard error of mean (s.e.m). Student’s unequal variances t-tests (two-tailed) of the data were performed to determine statistical significance of a positive protein-protein interaction using the OPCML-pM and RTK-pVP16 negative controls as baseline. All statistical analysis was carried out in Microsoft Excel.

Duolink® Proximity Ligation Assay (PLA) For PLA assay (Duolink®, OLink Biosciences, Sigma-Aldrich), the various cell lines were seeded on 13mm glass coverslips. After the required treatments, cells were fixed with ice-cold methanol for 5 minutes at -20˚C, washed with PBS and fixed with 3% BSA in PBS for 30 minutes at room temperature. Cells were then labelled with primary antibodies diluted 1:100 in 1% BSA/PBS at room temperature for 1 hour, taking care to use two different species (e.g. Mouse anti- HER2 and Rabbit anti-EGFR). Incubation with PLA probes MINUS and PLUS, ligation and amplification steps were carried out as per manufacturer’s instructions. Quantification of the number of Duolink® particles was performed using the ImageJ software.

Drug treatment and proliferation assay

Cells were seeded in triplicate in 96-well plates and treated with increasing concentrations of targeted therapy (0 – 100 PM) for 48 hours before measuring

cell viability by CellTiter 96® AQueous One Solution Cell Proliferation assay (Promega) following the manufacturer’s recommendations. GI50 values were calculated using the GraphPad Prism software. For western blot analysis, cells were treated with targeted therapy for 3 hours and then stimulated with 50 ng/ml rhEGF (R&D systems) for 30 min before the cells were lysed for 20 min on ice. Lapatinib (Sigma-Aldrich) and erlotinib (VWR) were reconstituted in dimethyl sulfoxide (DMSO) (Sigma-Aldrich) and used at the concentrations shown. Each experiment was repeated three times and a representative figure from each experiment is included in the result section.

Apoptosis assay Cells were seeded in triplicate in 96-well plates and treated with increasing concentrations of targeted therapy (0 – 120 PM) for 24 hours before assessing apoptosis by measuring caspase 3/7 activation using the Caspase-Glo® 3/7 assay (Promega) following manufacturer’s instructions. Caspase activity was

normalised to cell density obtained by CellTiter 96® AQueous One Solution Cell Proliferation assay (Promega) for each treatment. Each experiment was repeated three times.

Small Interference RNA (siRNA) SKOV3 cells were transfected in 24-well plates with 20nM of siRNA 24 hours post trypsinization. After cells were harvested, 2000 cells per well were seeded

® in triplicate in 96-well plates for measuring cell viability by CellTiter 96 AQueous One Solution Cell Proliferation assay (Promega) and 30000 cells per well were seeded in 24-well plate for western blot analysis. siRNAs containing a pool of siRNAs against EGFR2 (SMARTpool ON-TARGETplus L-003114) and HER2 (SMARTpool ON-TARGETplus L-003126) were transfected either alone or in combination, non-targeting siRNA pool #2 was used as control. The siRNAs were transfected using Oligofectamine (Invitrogen, Life Technologies Corporation, USA) in Opti-MEM media (GIBCO, Thermofisher Scientific) according to the

manufacturer’s protocol. All siRNAs were purchased from Dharmacon (GE Healthcare Life Science).

Immunohistochemical analysis Histopathological samples were obtained from 8 patients with advanced recurrent breast cancer that were treated previously with adjuvant chemotherapy and following treatment with lapatinib (Tyverb®). The following information was extracted from the histopathology reports: tumor type, grade, stage, estrogen receptor (ER), progesterone receptor (PR), as well as HER2 immuno-scores. Sections of 2 μm thickness from each tumor were cut from paraffin wax-embedded blocks, floated onto polysine slides, and dried overnight at 37 °C. The next day, sections were de-waxed, rehydrated and incubated with 0.6% peroxide block for 15 minutes to block endogenous peroxidase activity. Antigen retrieval was performed by microwaving the samples for 20 minutes in EDTA (pH 9.0) at 1,000W. Background staining was blocked using Power block (protein block supplied by Biogenex) for 10 minutes. The sections were then incubated with the monoclonal anti-human OPCML antibody (R&D Systems) at 1 μg/ml for 1 hour at room temperature and detected using Super-Sensitive Polymer-HRP detection kit with diaminobenzidine substrate at manufacturer’s instructions (Biogenex, Launch Diagnostics). Counterstaining was carried out with haematoxylin. For negative controls, duplicate sections were used in which the primary antibody was omitted and replaced with PBS. Sections were examined by light microscopy and staining was assessed with respect to presence, localization and intensity of staining. Staining was scored as weak (+), moderate (++) or strong (+++). Ethical approval to carry out expression analysis on snap-frozen samples from breast cancer patients was approved by Hammersmith and Queen Charlotte's & Chelsea Research Ethics Committee (REC reference 05/Q0406/178).

Results OPCML binds to HER2 but not EGFR, interfering with the formation of the HER2-EGFR heterodimer

Our previous co-immunoprecipitation (co-IP) experiments suggested that OPCML interacts with HER2, but not with EGFR, in the ovarian cancer cell line SKOV3 (9). Here we have investigated more in depth this difference by using a recombinant GST-OPCML to perform pull-down experiments in two breast cancer cell lines with no detectable OPCML expression: SKBR3, which overexpresses HER2 and is positive for EGFR, and MDA-MB-231, positive for EGFR and expressing very low levels of HER2. In SKBR3 cells, we found that HER2 was efficiently pulled-down in the cell lysate by GST-OPCML (Figure 1A), but no EGFR could be detected (Figure 1B), thus validating our previous findings. The pull-down assay was then repeated in the HER2-negative MDA- MB-231 breast cancer cell line and again no EGFR could be detected (Figure 1B), thus providing further evidence that OPCML does not interact with EGFR. In order to further confirm these results, we carried out mammalian 2-hybrid studies. The extracellular domains (ECD) of HER2 and EGFR were cloned to determine their ability to interact with OPCML (Figure S1) and transiently transfected in Cos-1 cells, which have a low basal expression of both HER2 and EGFR. The luminescence was normalised by co-transfection with a plasmid expressing the β-galactosidase gene and the empty pM and pVP16 vectors were used as negative controls. As presented in Figure 1C, a positive signal was obtained only when cells were co-transfected with OPCML and HER2, confirming again that OPCML interacts with HER2, but not with EGFR. Furthermore, we investigated which portion of the receptor was important for the binding to OPCML and deletion of domain 4 of the ECD of HER2 (HER2 '4, Figure S1) was shown to disrupt the interaction (Figure 1D). As the membrane- proximal portion of domain 4 of EGFR and HER2 has been proposed to mediate part of the interaction between the two molecules (37), we then analysed the ability of the two receptors to heterodimerize in the presence or absence of OPCML. In SKOV3 and SKBR3 cells, an ovarian and a breast cancer cell line, respectively, that express high levels of both HER2 and EGFR, HER2 was co- immunoprecipitated by an anti-EGFR antibody three times less in the presence of OPCML expression compared with OPCML non-expressing control cells (Figure 1E). We then confirmed these results further and quantified the interaction by using a proximity ligation assay (PLA). The Duolink® signal given

by the close vicinity of EGFR to HER2 in non-stimulated cells was maintained in control cells stimulated with EGF for 30 minutes and dropped after one hour stimulation, while in OPCML-expressing cells it was rapidly and strongly reduced by 30 minutes (Figure 1F). This suggests that OPCML interaction with HER2 induces a reduction of the HER2/EGFR dimer through dimer formation interference.

OPCML potentiates lapatinib and erlotinib therapy in ovarian and breast cancer cell lines overexpressing HER2 As we observed previously, OPCML binding to HER2 down-regulates the receptor and its downstream signaling in ovarian cancer cell lines (9). This led us to examine the potential therapeutic benefits of OPCML when combined with anti-HER2 agents. We first evaluated the impact of OPCML expression on 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 HER2 expression and with low or no HER3 and HER4 expression (Figure S2) were stably transduced to express OPCML (Figure S3) and treated with increasing amounts (0-100PM) of lapatinib for 48 hours before measuring their cell viability. In cells over-expressing HER2 (SKOV3 and SKBR3) we observed a significant decrease in cell viability when OPCML was present in

the cells (control GI50 = 34PM vs OPCML GI50 = 15.5PM for SKOV3, and control

GI50 = 13.6PM vs OPCML GI50 = 1PM in SKBR3), while in cells with little HER2 expression (OV90) the significance was lost even though a similar trend was maintained (Figure 2A). We then analysed the downstream signaling of these cell lines treated with serial dilutions of lapatinib for 3 hours followed by stimulation with EGF for 30 minutes. Lapatinib effects on the tyrosine phosphorylation levels of HER2 were very obvious at the lowest concentration of the drug (25nM) while phospho-EGFR levels were mostly inhibited at the highest concentrations (100-200nM) (Figure 2C-D), with the total levels of proteins being unaffected (Figure S4). These results confirm previous findings suggesting that lapatinib mainly acts through HER2 rather than EGFR (38). Overall, OPCML expressing cells had reduced amounts of the survival downstream effector phospho-AKT at lower concentrations of the drug compared with control cells in

SKOV3 and SKBR3 and, at a lesser level, in OV90 cells (Figure 2C-D), correlating

with the GI50 data. The effects of lapatinib on the Mitogen-Activated Protein Kinase (MAPK) pathway were also analysed but only OPCML-expressing SKOV3 cells showed reduction of the phospho-ERK1/2 signal compared with control cells (Figure 2C-D). The consistent downregulation of the phospho-AKT signal at lower doses of lapatinib mediated by OPCML in SKOV3 and SKBR3 cells was also reflected in activation of the apoptotic pathway in both cell lines. Caspase 3 and 7 activation, after 24 hours of treatment with lapatinib, was in fact strongly increased and detected at lower concentrations of the drug in the OPCML- expressing cells compared with control (Figure 2B). These results suggest that OPCML sensitizes cells to lapatinib mainly through pAKT inhibition- and caspase activation- pathway.

Since OPCML binds to HER2 making it less available for hetero-dimerization with EGFR (Figure 1), a therapy that targets only EGFR could still benefit from the presence of OPCML, even though OPCML does not affect EGFR levels directly. We investigated our hypothesis by treating the cells with the anti-EGFR kinase inhibitor erlotinib for growth inhibition studies and found that, similarly to what seen for lapatinib, SKOV3 and SKBR3 cells expressing OPCML had a significant decrease in cell viability compared with control cells, while in OV90 cells this difference was not apparent (Figure 3A). However, in OPCML-expressing cells

the degree of sensitization to erlotinib (Control GI50 = 32.5 PM and 24.3 PM vs

OPCML GI50 = 24.5 PM and 19.4 PM for SKOV3 and SKBR3 respectively) was much less compared with the degree of sensitization to lapatinib, highlighting that OPCML confers a more efficient sensitization of anti-HER2 therapy rather than anti-EGFR. Western blot analysis of the downstream signaling of these cell lines treated with erlotinib for 3 hours and stimulated with EGF for 30 minutes confirmed the cell viability results, in which the phospho-HER2 and phospho- AKT levels were inhibited at lower doses of erlotinib in OPCML-expressing SKOV3 and SKBR3 but not in OV90 cells (Figure 3C-D). Here, OPCML-mediated reduction of the phospho-ERK1/2 signal was found not only in SKOV3 cells but also, at a lower extent, in SKBR3 cells (Figure 3C-D). The ability of the SKBR3 cells to respond to EGFR-targeted therapy despite lack of downregulation of the

phospho-EGFR levels (Figure 3D) has also been found by others (39) and has been suggested as an evidence for sensitivity of HER2 overexpressing tumors to EGFR-specific TKIs (39–41). Caspase 3/7 activation assays in SKOV3 and SKBR3 cells treated with erlotinib for 24 hours confirmed the GI50 data and OPCML- expressing cells showed higher levels of the activated enzymes compared with control cells (Figure 3B) However, this was to a much lower extent compared with lapatinib treatment, suggesting again that the combined HER2-targeted therapy is more efficient in killing cancer cells.

These observations were further confirmed by genetic inhibition through small interference RNA (siRNA) of HER2, EGFR or both. When either HER2 or EGFR or both were silenced (Figure 4A), the viability of SKOV3 cells decreased significantly compared with a non-targeting control (siNT) after 48 and 72 hours from transfection (Figure 4B). However, OPCML-expressing cells showed a further decrease in cell viability at 72 hours compared with control cells when HER2, but not EGFR, protein levels were downregulated (Figure 4B).

Higher expression levels of OPCML correlate with better response to lapatinib in breast cancer patients and better survival in HER2 positive ovarian cancer patients In view of the above findings, we evaluated the predictive value of OPCML protein expression in a series of known HER2+ recurrent breast cancer patients receiving capecitabine and lapatinib. We had previously demonstrated that OPCML mRNA expression is a significant good prognostic factor for recurrence free survival in a series of over 2300 breast cancer patients (9). HER2+ (all cases 3+) breast cancer patients treated with capecitabine and lapatinib were selected (Figure S5). Following immunohistochemistry (IHC) staining for OPCML (Figure S6), this small series demonstrated a statistically significant correlation between OPCML protein expression and RECIST response to lapatinib/capecitabine (Fisher's Exact Test, two tailed p = 0.0286). All patients with 2+ and 3+ OPCML IHC expression responded, whilst none of the patients with 0 and 1+ OPCML IHC expression did (Figure S5).

We then looked at progression free survival (PFS) of ovarian cancer patients with HER2 overexpression (top quartile) in the Tothill dataset (42) and found that patients had a significantly better survival when OPCML expression was above median (log-rank p=0.004). In patients with low HER2 (bottom quartile) OPCML expression did not improve survival (Figure 5). Overall these data further confirm OPCML relevance in the regulation of HER2 signaling in a clinical setting.

Discussion OPCML is a protein that is frequently lost in tumors due to a combination of LOH and hypermethylation of the promoter of the gene (1,2). We previously described how its tumor suppressing function is achieved, showing that, by interacting with and downregulating a specific repertoire of RTKs, it causes a profound inhibition of the downstream ERK and AKT pathways (9). In particular, OPCML downregulates HER2 by changing the receptor localization on the cell membrane and inducing its internalisation through a non-clathrin mediated pathway (9). Here we have confirmed by pull-down and mammalian 2-hybrid assays the interaction between OPCML and HER2 and demonstrated by co-IP experiments and Duolink® assays that OPCML prevents the heterodimerization of HER2 with EGFR. We have also shown that the interference with the EGFR/HER2 heterodimer formation is mediated by the binding of OPCML to the domain 4 of the ECD of HER2, which is important for the stabilization of the heterodimer (37). Given these results, we have then investigated the ability of OPCML to sensitize cells to HER2- and EGFR-targeted therapies in HER2- overexpressing cancer cell models. By acting on HER2, OPCML affects the signaling from the EGF receptor family, as HER2 is the preferential partner of the three other members of the family (34,35), as it is always in an open conformation (43,44). We therefore used lapatinib (EGFR/HER2) and erlotinib (EGFR-only) RTK inhibitors in cancer cells bearing varying levels of expression of these two receptors (and no or little expression of HER3/4) in which we then re-expressed OPCML. Lapatinib acts as an ATP competitor for the kinase domain of both HER2 and EGFR causing the downregulation of the mitogenic and proliferative signaling driven by the activation of the two receptors (45). Despite

the promising initial response of patients to this drug, relapse and resistance are still common events. It has been then suggested that the combination of HER2- targeting therapies can be more effective in the initial line of treatment as well as in relapsing cancers (18). In this study we have shown how re-expression of OPCML in ovarian and breast cancer cell lines with high HER2 levels (SKOV3 and SKBR3) sensitizes these cells to lapatinib through caspase 3/7 activation. This confirms that by interfering with both HER2 dimerization (through OPCML interaction with the extracellular domain) and kinase activation (through lapatinib action) cells die at significantly lower concentrations of the inhibitor. The analysis of survival pathways mediated by AKT confirmed that cells expressing OPCML had a significant reduction in phospho-AKT levels at lower doses of lapatinib compared with control cells in both SKOV3 and SKBR3 cells and to a much lesser extent in OV90 cells, which have low expression levels of HER2. When treating the same cell lines with the EGFR inhibitor erlotinib we obtained similar results, with cells overexpressing HER2 being sensitized to the drug through a higher activation of caspase 3/7 and the phospho-AKT signaling being strongly affected by OPCML expression in SKOV3 and SKBR3 cells and only partially in OV90 cells. However, overall the magnitude of sensitization to erlotinib was lower compared with lapatinib, suggesting that OPCML targeting of HER2 is indeed a more efficient approach to inhibit cancer cell growth. These findings were also corroborated in a clinical setting, where breast cancer patients with HER2-positive tumors were found to be significantly more likely to respond to capecitabine/lapatinib therapy in the context of high tumoral OPCML protein expression levels compared with OPCML non/weakly expressing patients. Furthermore, stratification for HER2 and OPCML expression of ovarian cancer patients showed that higher co-expression of OPCML significantly increased progression free survival of HER2-overexpressing patients. Taken together these results show how, by acting through a key molecule such as HER2, OPCML amplifies the anticancer effect of lapatinib and to a lesser extent erlotinib and allows for a more effective targeting strategy of the EGFR-HER2 axis (Figure 6). Furthermore, these results support the notion of OPCML as a sensitizing agent in cancers with an activated HER2 status. Therefore we hypothesise that selection

by OPCML expression status might enrich for RTK inhibitor responders in HER2+ breast and ovarian cancer patients, although this requires further validation in the future. Additionally, we previously demonstrated that a recombinant OPCML biotherapeutic was an active anticancer agent preclinically in-vivo (9) and therefore this biotherapeutic could be used in combination with HER2 targeting TKIs to enhance their response. Considering that OPCML also downregulates other RTKs, these data also open up the possibility that this general effect of OPCML may apply to other RTK targeting scenarios, where different RTKs might be driving cancer growth. By acting at several levels in the RTK crosstalk, OPCML could delay or even prevent the onset of resistance that often follows the use of RTK inhibitors due to compensatory mechanisms. OPCML could therefore prove valuable as a therapeutic not just in RTK addicted cancers but also in cancers that are not strictly addicted to a specific oncogene, allowing responses that would not otherwise occur. (Word count: 4669)

Acknowledgements: We wish to acknowledge the support of the NIHR Biomedical Research Centre, the Cancer Research UK Clinical Centre and the Experimental Cancer Medicine Centre at Imperial College London.

References

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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 analysed by Western blot. GST only was used as a control. (C) The interaction between OPCML and the extracellular domains of HER2 and EGFR was detected by mammalian 2-hybrid. Luciferase activity was normalized to β-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 -hybrid. Luciferase activity was normalised to β-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 50ng/μl EGF for 0, 30 minutes or 1 hour. Duolink® particles were quantified using the ImageJ software.

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- 100μM) 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 (0-70μM for SKOV3 and 0- 120μM for SKBR3) of lapatinib for 24 hours. Caspase activity was normalised 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 50ng/ml of rhEGF for 30 minutes and the downstream signaling pathway was analysed by Western blot. (D) pHER2, pEGFR, pAKT and pERK1/2 signals obtained by densitometry analysis were normalized to calnexin and to untreated CTR cells (+/D). D: DMSO.

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- 100μM) 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 (0-96μM) of erlotinib for 24 hours. Caspase activity was normalised 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 50ng/ml of rhEGF for 30 minutes and the downstream signaling pathway was analysed by Western blot. (D) pHER2, pEGFR, pAKT and pERK1/2 signals obtained by densitometry analysis were normalized to calnexin and to untreated CTR cells (+/D). D: DMSO.

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 (siEG+siHE) 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.

Figure 5. High expression of OPCML in HER2-positive Ovarian Cancers is associated with better progression-free survival. Kaplan-Meyer survival

curves of 254 ovarian cancer patients from Tothill’s dataset based on OPCML (median) and HER2 (top and bottom quartiles) expression.

Figure 6. Schematic diagram of OPCML as a sensitizing agent. (A) Ligand binding to the extracellular domain 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.

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 Published OnlineFirst August 3, 2017.

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