Genomic Dissection of the Epidermal Growth Factor Receptor (EGFR)/PI3K Pathway Reveals Frequent Deletion of the EGFR Phosphatase PTPRS in Head and Neck Cancers

Genomic dissection of the epidermal growth factor receptor (EGFR)/PI3K pathway reveals frequent deletion of the EGFR phosphatase PTPRS in head and neck cancers

Luc G. T. Morrisa,b, Barry S. Taylorc, Trever G. Bivonaa, Yongxing Gonga, Stephanie Enga, Cameron W. Brennand,e, Andrew Kaufmana, Edward R. Kastenhubera, Victoria E. Banuchia, Bhuvanesh Singhb, Adriana Heguya, Agnes Vialef, Ingo K. Mellinghoffa,g, Jason Husea,h, Ian Ganlyb, and Timothy A. Chana,e,i,1

aHuman Oncology and Pathogenesis Program, Departments of bSurgery, dNeurosurgery, gNeurology, hPathology, and iRadiation Oncology, cComputational Biology Center, eBrain Tumor Center, and fGenomics Core, Memorial Sloan-Kettering Cancer Center, New York, NY 10065

Edited by Gregg L. Semenza, The Johns Hopkins University School of Medicine, Baltimore, MD, and approved October 13, 2011 (received for review July 23, 2011) Activation of the PI3K and epidermal growth factor receptor signiﬁcant promise in patients with head and neck cancers (21, (EGFR) pathway is able to drive oncogenesis in multiple human 22), but responses are heterogeneous and the genetic determi- cancers, including head and neck squamous cell carcinoma. nants of response are obscure. This lack of understanding is Targeted agents such as cetuximab and erlotinib are currently the primary factor hindering the effective use of these agents. used in patients with head and neck squamous cell carcinoma, In HNSCC, classical driver mutations of the pathway, such as but, in this disease, the genomic alterations that cause pathway PTEN and EGFR mutations, are rare, as are mutations in activation and determine response to pharmacologic inhibition ERBB2–4 and members of the RAS pathway (23–27). To resolve remain ill-deﬁned. Here, we present a detailed dissection of the this question, we undertook a detailed genomic dissection of EGFR/PI3K pathway, composed of sequencing of the core pathway the EGFR/PI3K pathway in oral cancer, the most common components, and high-resolution genomic copy number assess- subsite of HNSCC. Here, we describe the genetic landscape of ment. Mutations were found in PIK3CA (6%), but no point muta- this pathway in these cancers and identify a frequently altered

tions were observed in other pathway genes such as PTEN and modulator of sensitivity to EGFR inhibition. These findings have MEDICAL SCIENCES EGFR. In contrast, we observed frequent copy number alterations significant impact on our understanding of HNSCC oncogenesis of genes in the pathway, including PIK3CA, EGFR, protein tyrosine and facilitate the efficacious use of anti-EGFR/PI3K therapy. phosphatase receptor S (PTPRS), and RICTOR. In total, activating genetic pathway alterations were identified in 74% of head and Results and Discussion neck tumors. Importantly, intragenic microdeletions of the EGFR Copy Number Landscape of the EGFR/PI3K Pathway in HNSCC. The phosphatase PTPRS were frequent (26%), identifying this gene components of the EGFR/PI3K pathway have been well de- as a target of 19p13 loss. PTPRS loss promoted EGFR/PI3K pathway scribed. The pathway consists of 26 core gene products. To de- activation, modulated resistance to EGFR inhibition, and strongly termine whether these genes are genetically altered in HNSCC, determined survival in lung cancer patients with activating EGFR we used an integrated genetic strategy consisting of high-resolu- fi mutations. These ndings have important implications for our un- tion global copy number and mutational analysis of EGFR/PI3K derstanding of head and neck cancer tumorigenesis and for the pathway genes. We assembled a collection of 31 high-quality oral use of targeted agents for this malignancy. cavity HNSCC tumors (Table S1 and SI Materials and Methods). Tumor samples with matched normal tissue were collected during oral cancer | tumor suppressor routine clinical care with patient consent under an institutional review board-approved protocol and snap-frozen. Single-nucle- ead and neck squamous cell carcinoma (HNSCC) is the fifth otide polymorphism fingerprinting was used to ensure that all Hmost common cancer worldwide (1). As in several other tumor samples were obtained from unique patients and correctly cancers, activation of PI3K and epidermal growth factor receptor matched to normal tissue (28). To determine the copy number (EGFR) signaling HNSCC plays a key role in driving oncogen- landscape of HNSCC, DNA from microdissected tumors was esis (2–9). The high frequency of receptor tyrosine kinase/PI3K- extracted and analyzed using the Agilent 1M Human Compara- signaling activation has made this pathway an attractive target tive Genomic Hybridization (CGH) microarray (aCGH). for the development of novel therapeutic agents (10). However, Regions of recurrent copy number alteration (CNA) in in HNSCC, the nature of the genetic events underlying pathway HNSCC were identified using the RAE method (29), revealing a activation remains ill-defined. number of significant focal amplifications and deletions, as well When bound to ligand, EGFR activates the PI3Ks, leading to the phosphorylation of phosphatidylinositol on the 3′-hydroxyl group (11, 12). The resultant product, phosphatidylinositol-3,4,5- Author contributions: L.G.T.M. and T.A.C. designed research; L.G.T.M., B.S.T., T.G.B., Y.G., trisphosphate, activates AKT kinase (13, 14). Specific components S.E., A.K., E.R.K., V.E.B., and J.H. performed research; L.G.T.M., B.S., A.H., A.V., I.K.M., and of the EGFR/PI3K pathway have been found to be mutated in I.G. contributed new reagents/analytic tools; L.G.T.M., B.S.T., and C.W.B. analyzed data; human cancers, resulting in pathway activation. For example, and L.G.T.M. and T.A.C. wrote the paper. oncogenic mutations in EGFR, PIK3CA, and PTEN occur com- The authors declare no conflict of interest. monly in a variety of cancers, such as lung, breast, prostate cancer, This article is a PNAS Direct Submission. and glioblastoma (2, 7, 15–20). However, in many cancer types, Data deposition: The data reported in this paper have been deposited in the Gene Ex- including HNSCC, the primary genetic causes of pathway acti- pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo; Memorial Sloan-Kettering Cancer Center lung cancer dataset, http://cbio.mskcc.org/Public/lung_array_data; Duke vation are poorly understood. A comprehensive genetic analysis University lung cancer dataset, accession no. GSE3141; and head and neck cancer cell line would be useful in identifying the driving lesions underlying dataset, accession no. GSE21483. pathway activation in HNSCC. 1To whom correspondence should be addressed. E-mail: [email protected]. Furthermore, agents that target the EGFR/PI3K pathway, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. such as cetuximab, erlotinib, and PI3K inhibitors, have shown 1073/pnas.1111963108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1111963108 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 as gains and losses of 15 chromosomal arms (Fig. 1A and Table events in 19p13 including PTPRS are also counted, the frequency S2). The most frequent CNAs observed were gain of chomosome of significant PTPRS loss or deletion was 32% (11/31) (Fig. 1B). 3q, which harbors PIK3CA and is amplified in many cancers; loss To the best of our knowledge, focal somatic alteration of this of chromosome 3p, which harbors FHIT, TGFBR2, and FOXP1 gene has not been described in human disease. PTPRS has been and is frequently lost in epithelial cancers; and loss of chromo- shown to directly interact with and dephosphorylate EGFR (34, NKX3 DUSP4 – some 8p, which harbors and (30 33). EGFR/PI3K 35). We observed deletions in this gene that were highly focal, pathway genes within regions of significant copy number gain − robustly identifying PTPRS as the target of CNA on chromosome included PIK3CA (frequency = 45.2%, q = 7.45 × 10 7), RIC- − 19p13.3, a genomic region frequently lost in several cancers and TOR (frequency = 38.7%, q = 1.12 × 10 4), and EGFR (fre- − thought to harbor an as-yet-unidentified tumor suppressor (36– quency = 32.3%, q = 3.42 × 10 5). Pathway genes within 39). Interestingly, several unique tumors had similar deletions, significant areas of loss included PTPRS (frequency = 26%, q = − 3.42 × 10 5), which encodes a transmembrane tyrosine phos- suggesting that loss of these regions may be highly selected in phatase (Fig. 1B and Fig. S1). CNAs were confirmed by genomic HNSCC. Notably, however, one tumor had a deletion at exon 1, quantitative PCR (qPCR) (Fig. 1C). a region far removed from the other deletions. It is possible that clustering of copy number loss in the central region of the gene Protein Tyrosine Phosphatase Receptor S (PTPRS) Is Frequently results in more efficient elimination of the transcript. Alterna- Deleted in HNSCC. Of particular note, intragenic PTPRS deletion tively, it is also possible that the chromatin state of this region of was observed in 26% of samples (8/31). If broader deletion the gene makes it more susceptible to alteration. Hotspots for

Fig. 1. Genomic dissection of the EGFR/PI3K-signaling pathway identiﬁes alterations in the pathway, including frequent PTPRS deletion, in HNSCC. (A) Ge- nome-wide copy number aberrations in oral cancers. Ampliﬁcations (red) and deletions (blue) are indicated across the 22 autosomes by genomic coordinates. Regions with a false discovery rate (FDR) ≤10% are plotted, with chromosomes indicated at the center and centromeres in red. Analysis and scores were calculated as described (29). Genes of interest within targeted CNAs are shown above the peaks. Genes encoding proteins that are implicated in the EGFR/PI3K pathway are labeled in red. (B) Focal deletions of PTPRS in an aCGH segmentation map showing the region around the PTPRS gene. The genomic location along chromosome 19 is noted along the top. The color legend depicts the extent of copy number loss. PTPRS direction (arrows) and individual exons (thick blue bars) are labeled. (C) Validation of copy number alterations in HNSCC. Quantitative PCR was used to assess all copy number alterations detected by aCGH. Results for representative genes and tumors are shown. Each measurement was performed in triplicate. Error bars denote 1 SD.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1111963108 Morris et al. Downloaded by guest on September 26, 2021 DNA alterations are known to be influenced by chromatin evident from this analysis. First, the significant alterations were structure and features of the neighboring DNA sequence (40, all pathway activating, and the total frequency of activating 41). Our data here suggest that CNAs in these four components alterations in the EGFR/PI3K pathway was high (74%) in of the EGFR/PI3K pathway are important for pathway activation HNSCC. Second, it appears that genomic alterations that can in HNSCC, although we cannot definitively rule out involvement potentially increase PI3K pathway activity were not mutually of other genes within the regions of CNA. exclusive in HNSCC. This pattern has been observed in a number of malignancies, including cancers of the breast, endome- Genetic Alteration Within the PI3K Pathway Is Frequent in HNSCC. trium, and colon. For example, PIK3CA and PTEN mutations To determine whether components of the EGFR/PI3K pathway coexist in all three tumor types and, in many cancers, are the rule are altered by somatic point mutations, we sequenced the rather than the exception (43, 44). In HNSCC, the coexistence of coding exons of all 26 genes in the same tumors subjected to CNAs and mutations in EGFR/PI3K pathway genes occur be- copy number analysis (SI Materials and Methods and Table S3). cause these alterations may synergize to induce PI3K activity in PIK3CA mutations were observed (6%), but point mutations conditions of low ligand availability, or because the alterations were not found in other genes of the pathway, including EGFR are not entirely redundant (43). and PTEN (Fig. S2 and Table S4). Our high-resolution aCGH Unsupervised hierarchical clustering of CNAs revealed that data revealed no EGFRvIII deletions, consistent with some there are three subgroups of head and neck tumors defined by prior data in HNSCC (42). We integrated the data for mutations copy number cluster: those with minimal, intermediate, and high and significant CNAs within the EGFR/PI3K pathway genes levels of copy number alteration (Fig. 2B). Gain and loss of and categorized the alterations as either pathway-activating or PIK3CA, RICTOR, EGFR, and PTPRS are distributed across all pathway-inactivating (Fig. 2A and Fig. S2). Several patterns were groups, indicating that these CNAs are not solely a function of MEDICAL SCIENCES

Fig. 2. Copy number alterations in HNSCC promote EGFR/PI3K pathway activation and are associated with poorer prognosis. (A) Activating alterations of the EGFR/PI3K-signaling components were observed in 74% of cases. Alterations were defined as either mutations or significant CNAs and then classified as pathway activating (red) or inactivating (blue). Frequencies are indicated as a percentage of total cases (color bar). (B) Unsupervised hierarchical clustering of copy number alterations. (Upper) Copy number or mutation status of the listed genes is indicated as per the color legend. (Lower) Heat map in which red represents amplification, white represents copy neutral, and blue represents deletion. Dendrogram indicates that three groups of HNSCC exist as defined by copy number alteration patterns. (C) Functional activation of the PI3K pathway observed in tumors harboring pathway mutations or amplification. Tumors with the genotypes indicated are stained with the phospho-antibodies indicated at the left. Normal oral mucosa is used a control. (Scale bars, 500 μm.) (D) Disease-specific survival and (E) overall survival by phospho-Akt status. Immunohistochemistry was performed as described in SI Materials and Methods.(F) Disease-free survival by EGFR/PI3K genetic alteration status. Groups were compared using the log-rank test.

Morris et al. PNAS Early Edition | 3of6 Downloaded by guest on September 26, 2021 increasing genomic instability. To verify that tumors harboring EFGR, phospho-AKT, phospho-PRAS40, and phospho-S6, com- these genetic alterations had evidence of downstream pathway pared with matched normal tissue, indicating pathway activation. activation, we performed immunohistochemistry on these sam- (Fig. 3B). To determine whether PTPRS loss can directly promote ples using antibodies to phospho-AKT, phospho-PRAS40, and EGFR activation in HNSCC cancer cells, we used siRNA to knock phospho-S6. We found that tumors with EGFR/PI3K pathway down PTPRS in HNSCC cells. Depletion of PTPRS with two in- gene alterations were functionally activated by the signaling dependent siRNAs, but not with scrambled siRNA, led to dra- pathway (Fig. 2C). Both pathway activation and the presence matically increased phospho-EGFR, indicating that loss of the of genetic alteration within the pathway were associated with phosphatase is sufficient to promote EGFR activation (Fig. 3C). poorer prognosis in these patients (Fig. 2D). Consistent with these findings, we observed pathway activation (elevated levels of phospho-EGFR and phospho-AKT) in tumors PTPRS Loss Promotes EGFR Activation. Particularly intriguing is with PTPRS deletion, but not in tumors lacking PTPRS deletion the high frequency of focal PTPRS deletion. Alterations of and EGFR amplification (Fig. 3D). We then examined an in- PTPRS in cancer are poorly described, and no focal alterations dependent dataset of lung adenocarcinomas (45) and found that of this gene have been documented. Biochemical experiments low PTPRS expression correlated with increased levels of phospho- have demonstrated that PTPRS directly interacts with EGFR EGFR (P = 0.027) (Fig. S3). Combined with our observations and dephosphorylates and inactivates EGFR and loss of PTPRS above, these data suggest that PTPRS deletion in the absence of enhances EGFR-induced transformation (34, 35). The micro- EGFR gene alterations can promote EGFR/PI3K pathway activa- deletions in PTPRS result in loss of mRNA expression (Fig. 3A). tion in HNSCC. PTPRS deletions were observed along with PIK3CA or RICTOR alteration, but also occurred in the absence of any other pathway PTPRS Modulates Resistance to EGFR Inhibition. Although anti- alterations. Importantly, tumors with PTPRS deletion, but no EGFR agents such as cetuximab and erlotinib are used in the EGFR amplification or mutation, had substantial levels of phospho- treatment of HNSCC, responses to therapy are inconsistent, and

Fig. 3. Deletion of the PTPRS phosphatase activates EGFR. (A) Deletion of PTPRS results in loss of expression. Representative tumors with normal PTPRS copy number and eight tumors with PTPRS deletion (red) were examined for PTPRS mRNA levels using quantitative PCR. Error bars depict 1 SD. Normal (blue) denotes data from normal oral mucosa epithelial cells. Experiments were performed in triplicate. (B) Tumors with PTPRS deletion, but no EGFR alterations, had substantial amounts of phospho-EFGR, phospho-AKT, phospho-PRAS40, and phospho-S6, consistent with pathway activation. Pathway activation was not seen in matched normal tissue. (Scale bars, 500 μm.) (C) Knockdown of PTPRS increases levels of phospho-EGFR (pEGFR) in HNSCC cells. Knockdown was performed using two different siRNAs. Western blot was performed as described in SI Materials and Methods. NT, scrambled, nontargeting siRNA. (D) Tumors with PTPRS deletion demonstrate increased levels of phospho-EGFR and phospho-AKT, compared with tumors lacking PTPRS deletion or EGFR ampliﬁcation. (Scale bars, 100 μm.)

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1111963108 Morris et al. Downloaded by guest on September 26, 2021 the molecular determinants of response are still not well defined poorer outcome (P = 0.03) (Fig. 4D) (31), demonstrating that (22, 46, 47). Because PTPRS can directly regulate EGFR ac- PTPRS significantly influences clinical prognosis. tivity, we sought to determine whether PTPRS loss affects the These data demonstrate that components of the EGFR/PI3K sensitivity of cancer cells to EGFR inhibition. We performed cell pathway are frequently altered at the genetic level in HNSCC. viability assays using the tyrosine kinase inhibitor erlotinib, which This conclusion is consistent with previous reports examining clinically has activity in both HNSCC and lung cancer. We found a few specific candidates (49) and with some immunohisto- that PTPRS knockdown increased resistance of the EGFR-de- chemical studies (42). Our data elucidate the landscape of the pendent cell lines HCC827 (EGFR exon 19 deletion) and H3255 EGFR/PI3K pathway in HNSCC. Importantly, we observed that (EGFR L858R) to erlotinib treatment (Fig. 4A and Fig. S4). intragenic deletion of the EGFR phosphatase PTPRS is one of Similarly, knockdown of PTPRS in HNSCC cell lines sensitive to the most common events in HNSCC, helps promote EGFR/ fi EGFR inhibition signi cantly modulated response to erlotinib PI3K pathway activation, and modulates sensitivity to EGFR B (Fig. 4 and Fig. S4). inhibitors. These findings have important implications for the Like erlotinib, cetuximab also targets EGFR. We found that understanding of HNSCC oncogenesis and for the judicious use PTPRS expression predicted response to cetuximab in HNSCC of targeted agents in this malignancy. cell lines. Examination of an expression dataset of cetuximab- sensitive and cetuximab-resistant HNSCC cell lines showed that Materials and Methods PTPRS expression was significantly lower in resistant cells (P = × −4 C See SI Materials and Methods for a complete description of materials and- 6 10 )(Fig.4 ) (48). Unfortunately, expression datasets from methods. patients with tyrosine kinase inhibitor (TKI)-sensitive and -resistant HNSCC and lung tumors are not currently available for our anal- Tumor Samples, aCGH Analysis, and Bioinformatics. HNSCC tumors from the ysis. However, we analyzed outcomes in a clinical cohort of lung Memorial Sloan-Kettering Cancer Center were obtained after patient consent adenocarcinomas with TKI-sensitive EGFR mutations and found under an institutional review board-approved protocol. DNA was extracted that low PTPRS-expressing tumors were associated with markedly from microdissected tumors and assessed on the Agilent 1M comparative MEDICAL SCIENCES

Fig. 4. Loss of PTPRS promotes resistance to pharmacologic inhibition of EGFR. (A) Knockdown of PTPRS increases resistance to erlotinib in EGFR-mutant lung cancer cells (HCC827, H3255) and in (B) HNSCC cells (MDA-584, SCC-1, Tu-167), as demonstrated by a shift in half-maximal inhibitory concentrations. Cell viability experiments were performed six times. Error bars depict 1 SD. NT, scrambled, nontargeting siRNA. (C) PTPRS expression is signiﬁcantly lower in HNSCC cell lines resistant to cetuximab (as deﬁned in SI Materials and Methods). Each data point represents normalized expression data from three replicate microarrays of HNSCC cell lines (48). Boxplots represent medians and upper/lower quartiles. Groups were compared using a two-tailed t test. (D) Clinical outcome by PTPRS expression in lung adenocarcinomas with TKI-sensitive EGFR mutations. Cases treated at Memorial Sloan-Kettering Cancer Center were categorized aslow (below median) or normal PTPRS expressors, and recurrence and survival were calculated using the Kaplan–Meier method and a log-rank test (31).

Morris et al. PNAS Early Edition | 5of6 Downloaded by guest on September 26, 2021 genomic hybridization platform; copy number alterations were analyzed Joseph Nevins (Duke University) for help with the Duke Universitylung cancer using the RAE method (7, 29). expression dataset. B.S.T. is a David H. Koch Fellow in cancer genomics. This work was supported in part by The Louis Gerstner Foundation (to T.A.C.), the ACKNOWLEDGMENTS. We thank Kety Huberman, Igor Dolgalev, Sabrena Starr Cancer Consortium (to T.A.C.), The Geoffrey Beene Cancer Center (to Thomas, Olga Aminova, and Andrew Keyserian for excellent technical T.A.C.), The Doris Duke Charitable Foundation (to T.A.C.), the AVON assistance; Christine Chung (The Johns Hopkins University) for providing Foundation (to T.A.C.), the Flight Attendant’s Medical Research Institute (to SCC1 and Tu-167 cell lines and Esther P. Black (University of Kentucky) and T.A.C.), and National Institutes of Health Grant T32 CA009685 (to L.G.T.M.).

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