Loss of CPAP expression promotes sustained EGFR signaling and Epithelial-Mesenchymal Transition in oral cancer cells

Oral squamous cell carcinoma (OSCC) is the most common type of head and neck squamous cell carcinoma (HNSCC). Altered epidermal growth factor receptor (EGFR) levels can contribute to tumor metastasis and resistance to therapies. The epithelial-mesenchymal transition (EMT), by which epithelial cells acquire a mesenchymal and invasive phenotype, contributes significantly to tumor metastasis in OSCC, and EGFR signaling is known to promote this process. Microtubule inhibition therapies cause EGFR inactivation or increase the sensitivity to EGFR targeting drugs in various cancers including OSCC. In this study, using OSCC model, we show that loss of a microtubule/tubulin binding protein, centrosomal protein 4.1-associated protein (CPAP), which is critical for centriole biogenesis and normal functioning of centrosome, caused an increase in the EGFR levels and signaling and, enhanced the EMT features and invasiveness of OSCC cells. Further, depletion of CPAP increased the tumorigenicity of these cells in a xeno-transplant model. Importantly, CPAP loss-associated EMT features and invasiveness of multiple OSCC cells were attenuated upon depletion of EGFR in them. Overall, our novel observations suggest that in addition to its previously known regulatory role in centrosome biogenesis and function, CPAP plays an important role in suppressing EMT and tumorigenesis in OSCC by regulating EGFR homeostasis and signaling.


Introduction
Head and neck squamous cell carcinoma (HNSCC) represents the sixth most common cancer with more than 600,000 new patients diagnosed worldwide and it is linked to more than 300,000 deaths every year 1 . Most of the head and neck cancers are squamous cell carcinomas (HNSCC) that arise from mucosal surfaces of the oral cavity (OSCC). In most cancers including OSCC, altered epidermal growth factor receptor (EGFR/ErbB1/HER1) levels contribute to tumorigenesis, metastasis, and resistance to therapies and poor rate of patient survival [2][3][4][5][6] . The epithelialmesenchymal transition (EMT), by which epithelial cells acquire a mesenchymal and invasive phenotype, contributes significantly to these features 7,8 . EGFR is significantly altered in OSCC and its prolonged signaling is mitogenic, driving the uncontrolled proliferation of tumor cells 9,10 .
In addition, EGFR expression and its signal transduction pathways play an important role in determining the sensitivity to chemo or radiotherapy 11,12 . Importantly, excessive signaling through this receptor also triggers EMT in tumor cells, which makes them more invasive and metastatic, and resistant to chemotherapy [13][14][15][16] . Hence, this receptor has become one of the major targets for new therapies being investigated in OSCC 13,17,18 . Despite these advances in the understanding of EGFR signaling, the regulatory mechanisms underlying EGFR signaling and their effects on cancer initiation, progression and metastasis are not fully understood.
2% glutaraldehyde. Cells on the seeded membrane side were scraped off using a wet cotton swab, followed by staining of the membrane with 2% crystal violet stain. After numerous washes, filters were dried, membranes were cut and mounted on glass slides in immersion oil. Number of cells in various fields were imaged using 10X Omax objective and imaged using the Toupview software.
Average number of cells in various fields between 3 independent experiments has been plotted.
Tumor microarray analysis (TMA) patient tissues: Human tumor and adjacent normal tissues were collected from the Biorepository & Tissue Analysis at Hollings Cancer Centre and stained and scored blindly and imaged by the IHC core.
Tumor xenografts: Two million, doxycycline inducible Cal27/74B-shControl and -shCPAP were suspended in Matrigel to make it 50% concentration and injected subcutaneously in both the right and left flanks of 6-week-old male and female athymic nude mice. After sacrifice, tumors were removed and weighed. All animal experiments were approved by the Medical University of South Carolina Institutional Animal Care and Use Committee (Charleston, SC).

CPAP depletion endows OSCC cells with EMT phenotype and properties. While conducting
centriole biogenesis-related studies using HeLa cells, we observed that depletion, but not overexpression, of CPAP resulted in a typical EMT-like morphological change (not shown). Since OSCC is a highly prevalent cancer, we examined EMT features in OSCC cells upon CPAP depletion. Stable CPAP depletion was performed in 3 oral cancer cell-lines with: 1) an epithelial phenotype (SCC-Cal27), 2) a mesenchymal phenotype of primary tumor origin (UM-SCC-74A), and 3) a mesenchymal phenotype of recurrent tumor origin (UM-SCC-74B). Stable CPAPdepleted and control cell-lines were generated by transduction using pLKO.1-puro lentiviral vectors encoding CPAP shRNA and scrambled control shRNA respectively, followed by selection using puromycin. As observed in Fig. 1A, all three oral cancer cell-lines, with epithelial and mesenchymal phenotypes that were transduced with CPAP shRNA showed elongated morphology, a key feature of EMT, compared to control shRNA expressing cells. The spindle-like stretched appearance was more prominent in the CPAP-devoid mesenchymal cells. Immunoblot (IB) analysis of these cells revealed the elevated expression of one or more mesenchymal markers (vimentin, N-cadherin, Zeb and Slug) in all three cell types upon CPAP depletion compared to their control counterparts (Fig. 1B). Although SCC-Cal27 did not show detectable levels of vimentin, diminished levels of epithelial marker E-cadherin and higher expression of transcription factors Zeb and Slug were detected in these cells upon CPAP depletion. On the other hand, both UM-SCC-74A and UM-SCC-74B cells with CPAP depletion showed higher levels of mesenchymal markers vimentin, N-cadherin, Zeb1 and Slug. Expression levels of E-cadherin and vimentin were also examined by immunofluorescence microscopy. As observed in Fig. 1C, while CPAP-depleted SCC-Cal27 cells expressed relatively lower levels of E-cadherin, CPAP-depleted UM-SCC-74A and UM-SCC-74B cells expressed higher levels of vimentin compared to respective control cells. These observations suggest that loss of CPAP expression renders cells more susceptible to undergo EMT.
Since, cells undergoing the EMT process are known to be more invasive, control-shRNA and CPAP-shRNA expressing cells were subjected to cell migration and invasion assay using transwell inserts with and without matrigel coating. While the migratory properties of control and CPAP-depleted cells in membrane-well plates without matrigel coating were not different (not shown), all three OSCC cell lines with CPAP depletion showed significantly higher matrigel invasion ability compared to control cells (Fig. 1D). Collectively, these observations suggest that CPAP plays a role in inhibiting the EMT process in OSCC cells. into an athymic-nude mice. These mice were given doxycycline in drinking water and monitored for tumor growth at timely intervals. Tumor growth was relatively rapid (not shown) in CPAP-shRNA expressing, both SCC-Cal27 (epithelial) and UM-SCC-74B (mesenchymal), cell recipient groups compared to their control-shRNA recipient counterparts. Importantly, the average tumor weights upon euthanasia of SCC-Cal27 cell recipients on day 48 and UM-SCC-74B cell recipients on day 24 post-injection were significantly higher in CPAP-shRNA expressing cell recipients compared to respective controls (Fig. 1B). Of note, in our hands, in spite of the mesenchymal phenotype, UM-SCC-74A cells failed to induce tumor in mice even after 50 days post-injection (not shown). In a parallel experiment, a small cohort of mice received s.c. injection of UM-SCC-74B cells that overexpress CPAP under doxycycline inducible promoter and were monitored similarly. Tumor weights in mice that received CPAP overexpressing cells were relatively lower compared to control cell recipients (Supplemental Fig. 1). Overall, these results along with our in vitro findings demonstrating that CPAP knockdown enhances EMT properties and invasiveness ( Fig. 3) show that CPAP suppresses the tumorigenic properties of OSCC cells.

CPAP loss enhances the total and phospho-EGFR levels in OSCC cells. Since EGF treatment
induced EGFR signaling triggers EMT features in OSCC cells 63,69 , EGFR levels in CPAP depleted cells were examined. As observed in Fig. 3A, CPAP depletion resulted in a profound increase in the cellular EGFR protein levels in all the three tested OSCC cell-lines (SCC-Cal27, UM-SCC-74B and UM-SCC-74B) with either epithelial or mesenchymal phenotypes. Next, we treated these cells with EGF and examined for the cellular levels of phosphorylated EGFR levels, in protein equalized cell lysates, as an indication of active signaling by this receptor. Fig. 3B shows that, as expected, EGF treatment resulted in an increase in the levels of phosphorylated EGFR in control SCC-Cal27, UM-SCC-74B and UM-SCC-74B cells. Interestingly, the basal levels of phospho-EGFR were profoundly higher in CPAP-depleted cells to begin with, compared to control cells.
Further, the increase in phospho-EGFR levels upon EGF treatment appeared to be more rapid and striking in CPAP-depleted cells, particularly in SCC-Cal27 and UM-SCC-74A cells, compared to their control counterparts. Collectively, these observations suggest that CPAP has a crucial role in maintaining the homeostasis of growth factor receptors like EGFR and loss of CPAP results in, potentially, enhanced and persistent signaling through this receptor, causing EMT and enhancing the tumorigenic properties of OSCC cells.

EGFR activation under CPAP loss enhances EMT features of OSCC cells.
Since phospho-EGFR levels were higher in CPAP depleted OSCC cells and EGF treatment of these cells caused a rapid increase in this phospho-protein levels, the EMT features of untreated and EGF treated cells were examined. As observed in Fig. 3C, UM-SCC-74B cells with and without CPAP knockdown showed the classic spindle-like elongated EMT-associated morphology upon EGF treatment compared to respective control cells. While the untreated CPAP-depleted cells showed spontaneous EMT features (as mentioned in Fig. 2A), these morphologic changes were more pronounced upon EGF treatment. Examination of EMT associated markers in these cells revealed a profound upregulation of N-cadherin, but not Slug and Vimentin, levels in control cells upon EGF treatment. However, CPAP depleted cells showed, in addition to higher basal levels compared to control cells, an increase in the levels of N-cadherin, Slug and Vimentin upon EGFR treatment (Fig. 3D). Similarly, although the classic EMT associated elongated appearance was not observed with control epithelial SCC-Cal27 cells upon EGF treatment alone (not shown), Ecadherin levels were diminished in these control cells upon EGF treatment. Importantly, upon EGF treatment, an increase in the protein levels of Slug was observed only in CPAP depleted, but not control, SCC-Cal27 cells (Fig. 3E). These observations support the notion that enhanced EGFR signaling is responsible for the EMT-like features of CPAP depleted OSCC cells. and UM-SCC-74B) showed comparable migratory properties, based on the assay using trans-wells without matrigel, irrespective of CPAP and/or EGFR deficiency (Fig. 4B). However, while CPAP deficiency caused enhanced invasion by all three cell-lines in a matrigel transwell assay as per our observations of Fig.1, CPAP-deficient SCC-Cal27, UM-SCC-74B and UM-SCC-74B cells with EGFR depletion showed profoundly suppressed invasiveness compared to their counterparts without EGFR depletion (Fig. 4C). Overall, these observations confirm that CPAP loss associated EMT phenotype and enhanced tumorigenic properties of OSCC cells are EGFR-, and its enhanced signaling, -dependent.

Discussion
Here, using the OSCC model, we demonstrate a negative regulatory role for CPAP, an essential centriole biogenesis protein 54 , in tumor prevention by keeping the EGFR signaling and EMT at bay. CPAP is a microtubule and α-tubulin binding protein and the tubulin-binding property 49,74,75 is important for its function on the centrioles, especially in restricting the centriole length 28,45,52 . In addition, CPAP is required for spindle orientation, which defines normal and asymmetric cell divisions, abnormalities, which could lead to various clinical conditions including tumor malignancies 26,[52][53][54] . Recently, it has been shown that inhibition of CPAP-tubulin interaction prevents proliferation of centrosome-amplified cancer cells 62 . However, a role for CPAP as a tumor suppressor in regulating EGFR homeostasis and signaling, and EMT is not known. In this report, we show that although EGFR activation increases the cellular levels of CPAP, loss of CPAP not only increased the total and phosphorylated EGFR levels, but also EGFR dependent-EMT in OSCC. Our results shed light on a novel mechanism of tumor suppression by the centrioleassociated protein CPAP.
Centrosome amplification and higher expression of centrosomal proteins are key features of many cancers 62, 66-68 . However, whether these feature are the cause of OSCC and other cancers, or a consequence of tumor progression associated events is largely unknown. Tumor microenvironment is inflammatory and known to express higher levels of growth and cell transformation factors such as TGF1 and EGF, and/or their receptors [76][77][78][79][80] . In fact, our results also show that EGF and TGF treatments, which are known to promote the tumorigenic potential of cells, can not only induce EMT-like phenotype, but also increase the expression levels of CPAP in cancer cells. On the other hand, the observation that loss of CPAP results in enhanced EGFR levels and signaling, and EMT features in OSCC cells suggest that this microtubule/tubulin interacting protein negatively regulates the onset of EMT through promoting EGFR homeostasis.
EGFR overexpression was detected in majority of OSCC tumors and many other malignancies 81,82 . More than 90% of OSCC overexpress EGFR 83,84 . Associations have been made between the higher expression levels of EGFR and an aggressive phenotype, poor prognosis and resistance to anticancer therapy of OSCC 81  It has been shown that not only does EGF signaling promote EMT in OSCC cells 63,69 , but also that these cancer cells that are undergoing EMT express considerably lower levels of EGFR and are less susceptible to EGFR targeted therapies, which leads to their chemotherapeutic resistance 88 .
Interestingly, while CPAP depletion associated increase in total EGFR appears to be similar in three different cell lines tested, the dynamics of phosphorylated EGFR levels upon ligand activation appear to be different. Basal level of phospho-EGFR upon CPAP depletion was found to be profoundly higher in mesenchymal cell-line UM-SCC-74B which was derived from a recurrent metastatic tumor and in an OSCC cell line SCC-Cal27 with epithelial phenotype, compared to mesenchymal UM-SCC-74A which is of a primary metastatic tumor origin. However, while ligand activation caused the rapid induction of phospho-EGFR in CPAP depleted UM-SCC-74A, its levels appeared to be downregulated rapidly in UM-SCC-74B cells upon ligand activation in vitro. Further, ligand engagement associated increase in phospho-EGFR in CPAP depleted SCC-Cal27 was modest compared to UM-SCC-74A, albeit clearly higher than its respective control. These observations suggest that rapid tumor inducing property of UM-OSCC-74B could be due to low threshold activation and persistent signaling of EGFR in these cells, especially under CPAP deficiency. These dynamics could also explain why these three oral cell lines with different phenotypic and tumor inducing properties express EMT associated markers, Zeb and Slug particularly, differently under CPAP deficiency and upon EGFR depletion. Nevertheless, the common features of CPAP depleted OSCC cells are higher and persistent EGFR expression, enhanced invasiveness and/or tumorigenic ability. Importantly, results from our EGFR depletion studies show that these CPAP-deficiency associated effects are, in fact, EGFR-and, perhaps, its persistent signaling-dependent.
Overall, our observations, reveal that CPAP, a tubulin interacting protein which is critical for centriole biogenesis, has a preventive role in EMT and tumorigenesis by promoting EGFR homeostasis and diminishing the EGFR signaling. While it has been reported before that EGF stimulation endows OSCC cells with stem cell-like properties, increased invasiveness, and tumorigenic properties 63,69 , the molecular mechanisms underlying the regulation of EGF induced EMT and tumorigenicity were not known. Further, although many studies have shown that microtubule inhibition causes EGFR inactivation or increases the sensitivity to EGFR targeting drugs in various cancers including OSCC [19][20][21][22] , whether centrosomes and centriolar proteins contribute to this effect was not investigated before. Hence, this study begins to shed light on the molecular mechanisms by which centrosome/MTOC associated proteins are involved in preventing tumorigenesis.