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Biology of Human Tumors

MUC16 Regulates TSPYL5 for Lung Cancer Cell Growth and Chemoresistance by Suppressing p53

Imayavaramban Lakshmanan, Shereen Salfity, Parthasarathy Seshacharyulu, Satyanarayana Rachagani, Abigail Thomas, Srustidhar Das, Prabin D. Majhi, Rama Krishna Nimmakayala, Raghupathy Vengoji, Subodh M. Lele, Moorthy P. Ponnusamy, Surinder K. Batra and Apar Kishor Ganti
DOI: 10.1158/1078-0432.CCR-16-2530 Published July 2017

Abstract

Purpose: MUC16, a tumor biomarker and cell surface–associated mucin, is overexpressed in various cancers; however, its role in lung cancer pathogenesis is unknown. Here, we have explored the mechanistic role of MUC16 in lung cancer.

Experimental Design: To identify the functional role of MUC16, stable knockdown was carried in lung cancer cells with two different shRNAs. Clinical significance of MUC16 was evaluated in lung cancer patient tissues using IHC. We have generated genetically engineered mouse model (KrasG12D; AdCre) to evaluate the preclinical significance of MUC16.

Results: MUC16 was overexpressed (P = 0.03) in lung cancer as compared with normal tissues. MUC16 knockdown (KD) in lung cancer cell lines decreased the in vitro growth rate (P < 0.05), migration (P < 0.001), and in vivo tumor growth (P = 0.007), whereas overexpression of MUC16-carboxyl terminal (MUC16-Cter) resulted in increased growth rate (P < 0.001). Transcriptome analysis of MUC16 KD showed a downregulation (P = 0.005) of TSPYL5 gene, which encodes for a testis-specific Y-like protein. Rescue studies via overexpression of MUC16-Cter in MUC16 KD cells showed activation of signaling proteins, such as JAK2 (Y1007/1008), STAT3 (Y705), and glucocorticoid receptor (GR), which constitutes an important axis for the regulation of TSPYL5 for oncogenic process. Further, inhibition of STAT3 (Y705) led to decreased GR and TSPYL5, suggesting that MUC16 regulates TSPYL5 through the JAK2/STAT3/GR axis. Also, MUC16 overexpression induced cisplatin and gemcitabine resistance by downregulation of p53.

Conclusions: Our findings indicate a significant role of MUC16 in tumorigenesis and metastasis of lung cancer cells possibly via regulation of TSPYL5 through the JAK2/STAT3/GR axis. Clin Cancer Res; 23(14); 3906–17. ©2017 AACR.

Translational Relevance

Although MUC16 has been shown to be involved in the growth and metastasis of several cancers, its role in lung carcinoma remains unclear. Herein, we have shown subtype-specific expression of MUC16 in lung adenocarcinoma. MUC16 expression seems to increase the aggressiveness of lung cancer cells. In addition, MUC16 appears to mediate chemoresistance via expression of TSPYL5 and consequent inactivation of p53 (wild-type) in lung cancer. Targeting the MUC16/TSPYL5 pathway may help in decreasing the aggressiveness and metastatic potential of lung cancer cells and in overcoming chemoresistance, thereby improving outcomes.

Introduction

MUC16 mucin is a large-molecular-weight (20 to 25 mD) glycoprotein with 22,152 amino acid (aa) residues in its protein sequence (1–3). MUC16 is a type I transmembrane protein that has three major domains: highly O-glycosylated N-terminal domain, repetitive sea urchin sperm, enterokinase and agrin (SEA) containing tandem repeat domain, and a cytoplasmic domain with potential phosphorylation sites (3, 4). The N-terminal portion of MUC16 interacts with mesothelin that facilitates the peritoneal metastasis of ovarian cancer cells (5, 6). The SEA domain of the tandem repeat region is responsible for the cleavage process (7), whereas carboxyl-terminal region contains 32 aa comprising of three tyrosine, two threonine, and one serine residues, which serve as potential phosphorylation sites for intracellular signaling (8, 9).

MUC16 is overexpressed and associated with poor prognosis in ovarian (10), breast (11), and pancreatic cancer (12). MUC16 is elevated in patients with multiple brain metastases from non–small cell lung cancer (NSCLC), and it is associated with poor prognosis (13). Another study has shown that MUC16 is elevated in stage I NSCLC patients' serum samples and demonstrated that MUC16 could be a useful biomarker for patients with lung cancer (14). Recent studies have reported that MUC16 is an extremely highly mutated gene, in various cancers including lung cancer (15). MUC16 has been shown to be associated with enhanced cancer cell growth and metastasis (4, 8). During this process, MUC16 interacts with various proteins such as mesothelin (5), JAK2 (11), and Src (8), and their association facilitates cancer cell growth and metastasis.

Due to its large size, several studies have been conducted with a small portion of the carboxyl terminal (Cter) of MUC16 (344 aa and 114 aa; refs. 4, 8, 16). It has been reported that MUC16-Cter has a strong oncogenic role in ovarian (8) and pancreatic cancers (4). However, the mechanistic and functional role of MUC16 in lung cancer is not well understood.

Testis-specific Y-like protein 5 (TSPYL5) gene is located at chromosome 8q22 (17); it has been frequently amplified in breast cancer and is associated with a poor prognosis (18). Epping and colleagues have demonstrated that TSPYL5 interacts with ubiquitin-specific protease 7 (USP7) that facilitates p53 degradation to suppress the tumor-suppressor activity of p53 (17). TSPYL5 has been shown to be involved in cancer cell growth by activating Akt signaling and was found to be involved in radioresistance in lung cancer cells (19). The nuclear hormone receptor family protein, glucocorticoid receptor (GR), is overexpressed in lung cancer and promotes cancer cell proliferation (20). Ligand (glucocorticoid) binding to GR leads to translocation of GR from cytoplasm to nucleus, where it directly binds to DNA and is involved in gene regulation (21). In this study, we evaluated the role of MUC16 in the growth, proliferation, spread, and chemosensitivity of lung cancer.

Materials and Methods

Cell culture and transfection

H292, H1975, and A549 lung cancer cells were cultured in RPMI medium supplemented with 10% FBS and antibiotics. The cell lines used in this study were recently obtained from the ATCC and revived from early-passage –140 freezer stocks. Cells were routinely inspected for phenotypic variation and mycoplasma contamination. Similarly, mouse tumor cell line K1418 was also cultured in DMEM medium with the above-mentioned supplements. The cells were incubated in a humidified atmosphere at 37°C with 5% CO2. Human-specific MUC16-shRNA (pSUPER-Retro-shMUC16 seq1 and pSUPER-Retro-shMUC16 seq2) and mouse-specific pSUPER-Retro-shmuc16 constructs were used for stable transfection of MUC16 in H292, H1975, and K1418 with respective control shRNA (4, 22).

Generation of spontaneous lung cancer mouse model

Genetically engineered mouse models LSL-KrasG12D (B6.129-Krastm4Tyj (01XJ6)) were developed by the Tuveson lab (23). Animals that were positive for KrasG12D were infected with AdCre-Luciferase retroviral vector intranasally (University of Iowa, Gene and vector core, Iowa). Eight weeks after infection, the animals were injected with luciferin intraperitoneally to monitor the tumor growth (22). Mice were fed with food and water ad libitum and subjected to a 12-hour light/dark cycle. The mice studies were performed in accordance with the U.S. Public Health Service “Guidelines for the Care and Use of Laboratory Animals” under an approved protocol by the Institutional Animal Care and Use Committee of the University of Nebraska Medical Center (UNMC). The mouse tumor tissues were utilized for immunostaining as described previously (24).

TMA and immunohistochemistry

The clinical specimen for IHC was a commercial tissues microarray (TMA; LC121 and LC 814; US Biomax). The LC121 included 120 cases of various histologic types of lung carcinoma [squamous cell carcinoma (n = 20), large cell carcinoma (n = 37), adenocarcinoma (n = 44), and normal lung tissues (n = 10)]. Similarly, LC814 included 40 cases of lung carcinoma (n = 40) and metastatic lymph node carcinoma (n = 40). The TMA was analyzed for MUC16 expression by IHC as described previously (24).

Immunoblot analysis

Western blot assay was performed as described previously (24). The blots were incubated with following primary antibodies with respective dilutions: MUC16 (mouse, 1:1,000), MUC16 (mouse, 1:1,000), pJAK2 #8082, JAK2 #3230, pSTAT3 #9145, STAT3 #12640, GR #12041, pSrc #2101 (Rabbit, 1:2,000; Cell Signaling Technology), E-cadherin (mouse, 1:1,500), and N-cadherin (mouse, 1:1,500) antibodies were a kind gift from Dr. Keith R Johnson, UNMC, Omaha, NE; CK-18 (mouse, 1:1,500; Abcam #668), TSPYL5 (rabbit 1:500; Santa Cruz Biotechnology, #sc-98185), p53 (mouse 1:500; Santa Cruz Biotechnology, #sc-126), and anti–β-actin (mouse 1:5,000; Sigma #A1978, diluted in 2% BSA in PBS). Similarly, immunoprecipitation assay was performed as described previously (22). The signals were detected with the ECL chemiluminescence Kit (Amersham Bioscience).

Quantitative real-time PCR, growth kinetics, transwell migration, and wound-healing assay

qPCR, growth kinetics, transwell migration, and wound-healing assays were performed as described previously (11, 24).

Phosphorylation-specific JAK and STAT3 inhibition

Ruxolitinib (1 μmol/L and 5 μmol/L) and phospho-specific STAT3 (Y705) Inhibitor XIII, C188-9 (5 μmol/L and 10 μmol/L), were used to confirm the MUC16/JAK2/STAT3 downstream signaling pathway in lung cancer cells. MUC16 knockdown and MUC16-Cter–overexpressed cells were treated with a different concentration of ruxolitinib and C188-9 for 24 hours; for control, 0.01% DMSO was used.

MTT assay

The cell viability of cisplatin- and gemcitabine-treated lung cancer cells was determined using MTT assay as described previously (24).

Long-term cisplatin treatment of lung cancer cells

We have generated the cisplatin-resistant cell line H292 by continuous incubation of lung cancer cells with cisplatin as described previously with slight modification (25). H292 cells were continuously treated with an increasing dose of cisplatin (100 nmol/L, 200 nmol/L, 400 nmol/L, 800 nmol/L, 1,600 nmol/L, and 3600 nmol/L) for 5 days/week for 12 weeks and leaving 2 days off for recovery. After 12 weeks of cisplatin treatment, the H292 cells were used for further experiments.

Data analysis

Statistical significance was evaluated with the Student t test using sigmaPlot 11.0 software. P values 00.05 were considered to be significant. Densitometry analyses were performed using ImageJ software for wound-healing experiments. All experiments were performed in triplicates.

Results

Expression of MUC16 in lung carcinoma

To investigate the clinical significance of MUC16 in patients with lung cancer, we examined the expression of MUC16 in the normal lung (n = 10) and human lung cancer tissues (n = 101). MUC16 was significantly overexpressed (P = 0.03) in human lung carcinoma (Fig. 1A) compared with normal lung. Among the NSCLC subtypes, MUC16 was detected in a higher proportion of adenocarcinoma (19/44, 43%) as compared with the squamous (1/20, 5%) and large cell carcinoma (11/37, 29.7%; P < 0.0001 for the comparison between adenocarcinoma and squamous cell carcinoma; Supplementary Fig. S1A). These are similar to the findings of The Cancer Genome Atlas database wherein MUC16 is overexpressed in a greater proportion of lung adenocarcinoma tissues compared with squamous cell carcinoma (Supplementary Fig. S1B). Furthermore, patients with high MUC16-expressing lung tumors had worse survival compared with patients who had low MUC16 expression (Fig. 1B) as demonstrated by the Kaplan–Meier curves (26). We also analyzed a commercial tissue array containing primary lung carcinoma (n = 40) and corresponding metastatic lymph node patient tissues (n = 40). In addition to detection of MUC16 in primary lung carcinoma, we also observed in lymph node metastases (Fig. 1C). We have generated a heat map to compare intensity of MUC16 in primary lung carcinoma and metastatic lymph node tissues using composite score (Fig. 1D). Muc16 was strongly overexpressed in mouse lung adenocarcinoma (KrasG12D; AdCre) as compared with normal bronchial tissues (Fig. 1E).

Figure 1.
Figure 1.

MUC16 expression in lung carcinoma and association of MUC16 with lung cancer patient survival. A, MUC16 was observed in a smaller proportion of normal bronchial tissues (1/10) but in a higher proportion of lung carcinoma (31/101, 30.69%; P = 0.03). B, MUC16 expression is associated with worse outcomes in patients with lung cancer. C, MUC16 expression was retained in both primary and metastatic lymph node tissues. D, Heat map of composite score represents that MUC16 expression in both primary lung carcinoma and matched lymph node tissues. E, Immunohistochemical results show that Muc16 is strongly overexpressed in mouse lung adenocarcinoma tissues (KrasG12D; AdCre) as compared with littermate control lung tissues (KrasG12D). *, P < 0.05. A, C, and E, magnification, X20; A, lower right, higher magnification, X40.

Stable knockdown of MUC16 in human lung cancer cells (H292 and H1975) and overexpression of MUC16-Cter in A549 lung cancer cells

In order to identify the functional role of MUC16 in lung cancer, we performed stable knockdown of MUC16 (two different shRNA targets) in two human lung cancer cell lines H292 and H1975 (Fig. 2A and B). In order to examine the function of the cytoplasmic tail region of MUC16 (MUC16-Cter) on lung cancer cells, we ectopically overexpressed MUC16-Cter (F114HA) in the MUC16-negative cell line A549 (Fig. 2C).

Figure 2.
Figure 2.

Stable of knockdown of MUC16 and ectopic overexpression of MUC16-Cter in lung cancer cells and its role in lung cancer cell growth and tumorigenicity. A and B, MUC16 is endogenously present in both H292 and H1975 lung cancer cells, and its expression was silenced using the pSUPER-Retro shRNA method with two different targets (shMUC16 seq1 and shMUC16 seq2). D and E, The growth of MUC16 knockdown cells (shMUC16 seq1 and shMUC16 seq2) was significantly (P < 0.05) reduced. C, We ectopically overexpressed MUC16-Cter (F114HA) in MUC16-negative lung cancer cell A549. F, MUC16-Cter–overexpressed lung cancer cells (A549-F114HA) had a higher growth rate (P < 0.05) than vector-transfected (A549-CMV9) cells. G, We performed tumorigenic assay by subcutaneously injecting MUC16 knockdown and scramble in athymic mice. MUC16 knockdown cells [H292-shMUC16 seq1 (P = 0.007) and seq2 (P = 0.04)] had significantly less tumorigenic capacity than scramble (H292-SCR) cells. H, MUC16 expression was low in tumors induced by MUC16 knockdown (H292-shMUC16 seq1 and seq2) cells as compared with scramble (H292-SCR) cells. β-Actin was used as loading control. *, P < 0.05; **, P < 0.01; ***, P < 0.001; and NS, nonsignificant. H, Magnification, X20.

Effect of MUC16 on lung cancer cell growth

The growth rate of MUC16 knockdown cells (H292-shMUC16 seq1 and seq2 and H1975-shMUC16 seq1 and seq2) was significantly decreased (P < 0.05) compared with scramble (H292-SCR and H1975-SCR) cells in growth kinetics assays (Fig. 2D and E). Similarly, growth kinetics assays showed that MUC16-Cter–overexpressed A549 (A549-F114HA) cells had significantly higher growth rate compared with vector cells (A549-CMV9; P < 0.05; Fig. 2F). This result indicates that MUC16 might play a crucial role in lung cancer cell growth.

Role of MUC16 on tumorigenicity of lung cancer cells

MUC16 knockdown (H292-shMUC16 seq1 and seq2) and scramble (H292-SCR) cells were subcutaneously implanted in the right flank region of the athymic nude mice. After 4 weeks, mice were sacrificed, and tumor weight was analyzed. MUC16 knockdown (H292-shMUC16 seq1 and seq2) cells had a significantly smaller tumor volume than scramble (H292-SCR) cells (P = 0.007 and P = 0.04, respectively; Fig. 2G). Analysis of MUC16 expression in xenograft tissues by IHC confirmed that MUC16 expression was decreased in tumors from knockdown (H292-shMUC16 seq1 and seq2) cells as compared with scramble (H292-SCR) cells (Fig. 2H).

MUC16 induces lung cancer cell migration through epithelial-to-mesenchymal transition

Transwell migration assay showed that MUC16 knockdown (H292-shMUC16 seq1 and seq2) cells have a decreased migratory capacity (P < 0.001; Fig. 3A). On the other hand, MUC16-Cter–overexpressed (A549-F114HA) cells had increased migratory capacity than vector (A549-CMV9) cells (Fig. 3B). Wound-healing assays demonstrated that the migration of MUC16 knockdown (H292-shMUC16 seq1 and seq2) cells was significantly reduced as compared with scramble (H292-SCR) cells (Fig. 3C and D).

Figure 3.
Figure 3.

Effect of MUC16 on the migration of lung cancer cells. A, Transwell migration assay demonstrates that migration of MUC16 knockdown [H292-shMUC16 seq1 (P = 0.001) and seq2 (P = 0.0006)] cells was significantly decreased. B, Similarly, MUC16-Cter–overexpressed cells have more migratory (P = 0.02) capacity as compared with vector cells. C, MUC16 knockdown (H292-shMUC16 seq1 and seq2) cells have less migratory capacity than scramble (H292-SCR) cells as demonstrated by a wound-healing assay. The wound area was stained with crystal violet for better visualization. D, The area was quantitatively calculated and normalized with wound area at 0 hours of respective controls. E, The phosphorylation of Src (Y416) was decreased in MUC16 knockdown (H292-shMUC16 seq1 and seq2). E, Expression of mesenchymal marker N-cadherin was decreased in MUC16 knockdown (H292-shMUC16 seq1 and seq2) cells, whereas epithelial marker CK-18 was increased. F, Increased phosphorylation of Src (Y416), increased expression of N-cadherin, and downregulation of CK18 were observed in MUC16-Cter overexpressed (A549-F114HA). β-Actin was used as loading control. *, P < 0.05; **, P < 0.01; and ***, P < 0.001. A, B, and C, Magnification, X10.

Phosphorylation of Src (Y416) was decreased in MUC16 knockdown cells (Fig. 3E). Similarly, the mesenchymal marker N-cadherin was decreased upon MUC16 knockdown, whereas epithelial marker CK18 was increased (Fig. 3E). MUC16-Cter–overexpressed cells showed increased phosphorylation of Src (Y416), increased expression of N-cadherin, and decreased CK-18 (Fig. 3F). These results suggest that MUC16 may have a role in the migration of lung cancer cells, possibly through Src signaling.

MUC16 downregulates TSPYL5 in lung cancer

A microarray analysis performed to analyze the MUC16-associated and -regulated genes in lung cancer revealed that TSPYL5 was significantly downregulated in MUC16 knockdown cells (P = 0.005). To validate the microarray data, we confirmed the TSPYL5 downregulation in MUC16 knockdown cells by real-time PCR analysis (Fig. 4A; Supplementary Fig. S1C). Similarly, MUC16-Cter–overexpressed cells have increased expression of TSPYL5 than vector cells (Fig. 4B).

Figure 4.
Figure 4.

MUC16 mediated downstream oncogenic signaling and inhibition of the JAK2/STAT3 pathway for TSPYL5 gene expression. A, Upon MUC16 knockdown, phosphorylation of JAK2 (Y1007/1008) and STAT3 (Y705) was decreased. B, Similarly, MUC16-Cter–overexpressed cells also had increased phosphorylation of JAK2 (Y1007/1008) and STAT3 (Y705). A and B, Similarly, GR and TSPYL5 were decreased in MUC16 knockdown and/or increased in MUC16-Cter–overexpressed lung cancer cells, respectively. C, We inhibited STAT3 (Y705) using specific inhibitor XIII, C188-9, in lung cancer. Phosphorylation of STAT3 (Y705) was decreased following pharmacologic inhibition of STAT3 (Y705) using two different concentrations (5 μmol/L and 10 μmol/L) of C188-9. C, Total STAT3 expression remained the same. Further, as a result of STAT3 (Y705) inhibition, GR and TSPYL5 were decreased as compared with untreated cells. D, Inhibition of phospho-STAT3 inhibition in MUC16-Cter–overexpressed cells confirmed the above findings. β-Actin was used as loading control.

MUC16 regulates TSPYL5 through JAK/STAT3/GR pathways

Phosphorylation of JAK2 (Y1007/1008) was decreased in MUC16 knockdown cells (H292-shMUC16 seq1 and seq2; Fig. 4A). Similarly, phosphorylation of STAT3 (Y705) was also decreased in MUC16 knockdown cells (Fig. 4A). On the other hand, MUC16-Cter–overexpressed lung cancer cells (A549-F114HA) showed increased phosphorylation of JAK2 (Y1007/1008) and STAT3 (Y705) as compared with vector cells (A549-CMV9; Fig. 4B). These results suggest that MUC16 stimulates JAK2/STAT3 signaling pathways for lung cancer cell growth. In addition, expression of the GR was decreased in MUC16 knockdown cells (Fig. 4A) as compared with scramble cells. Similarly, MUC16-Cter–overexpressed (A549-F114HA) cells had a higher level of GR than vector cells (Fig. 4B).

TSPYL5 promoter studies show that promoter region of TSPYL5 has GR-binding sites (Biobase software). Furthermore, STAT3 and GR act synergistically for regulation of various subsets of genes including Fox, CREB, and AP-1 (20, 27, 28). Here, we observed an interaction between STAT3 and GR in lung cancer by immunoprecipitation assay (Supplementary Fig. S1D). Based on this finding, we suggest that MUC16 regulates the transcription factors STAT3 and GR eventually affecting TSPYL5 gene expression.

Inhibition of JAK2 and STAT3 in lung cancer cells

We inhibited JAK1/2 (ruxolitinib; refs. 29, 30) in H292 cells. Following JAK1/2 inhibition, we analyzed the tyrosine phosphorylation of STAT3 (Y705), which is the downstream signaling target of JAK2 (31). Tyrosine phosphorylation of STAT3 (Y705) was decreased in JAK1/2 inhibitor (1 μmol/L and 5 μmol/L)–treated cells (Supplementary Fig. S2A). Similarly, we also inhibited STAT3 (Y705) phosphorylation by STAT3 (Y705)-specific Inhibitor XIII, C188-9 (32), at two different concentrations: 5 μmol/L and 10 μmol/L (32). Following STAT3 inhibition, we observed the decreased phosphorylation of STAT3 (Y705; Fig. 4C). Upon STAT3 (Y705) inhibition, we observed a decreased expression of GR and TSPYL5 (Fig. 4C).

Similar experiments were also performed in MUC16-Cter overexpressed in A549 cells. Following JAK1/2 inhibition, we observed decreased phosphorylation of STAT3 (Y705; Supplementary Fig. S2B). In addition, STAT3 (Y705) inhibition in MUC16-Cter–overexpressed cells resulted in decreased GR and TSPYL5 as compared with untreated cells (Fig. 4D). These results indicate that MUC16-mediated JAK2/STAT3/GR signaling leads to TSPYL5 gene regulation, which in turn may cause lung cancer cell growth and metastasis.

MUC16 contributes to cisplatin and gemcitabine resistance

MUC16 knockdown cells (H292-shMUC16 seq1 and seq 2, H1975-shMUC16 seq1 and seq 2) were more sensitive to cisplatin (Fig. 5A; Supplementary Fig. S3A) and gemcitabine (Fig. 5B; Supplementary Fig. S3B) as demonstrated by the MTT assay. Upon cisplatin treatment, MUC16 knockdown cells had higher apoptosis (Supplementary Fig. S3C). In contrast, no significant change was observed in the untreated scramble (H292-SCR) and MUC16 knockdown (H292-shMUC16 seq1 and shMUC16 seq2) cells (Supplementary Fig. S3C). Similarly, MUC16-Cter–overexpressed lung cancer cells (A549-F114HA) were more resistant to the cytotoxic effects of cisplatin (Fig. 5C) and gemcitabine (Fig. 5D).

Figure 5.
Figure 5.

Role of MUC16 in cisplatin and gemcitabine resistance. A and B, We treated MUC16 knockdown (H292-shMUC16 seq1 and seq2) and scramble (H292-SCR) cells with various concentrations of cisplatin and gemcitabine. MTT assay results show that MUC16 knockdown (P < 0.05) cells were more responsive to cisplatin (A) and gemcitabine (B). C and D, Similarly, MUC16-Cter–overexpressed cells (P < 0.05) were more resistant to cisplatin (C) and gemcitabine (D). E and F, Muc16 knockdown (K1418-shMuc16) genetically engineered mouse model (GEMM) tumor cells (P < 0.05) were more sensitive to cisplatin (E) and gemcitabine (F) than scramble cells. Mechanism of MUC16 mediated chemoresistance. G, Upon MUC16 knockdown, expression of p53 was increased as compared with scramble. H, The expression of p53 was high in tumors derived by subcutaneous injection of MUC16 knockdown cells as compared with tumors derived by injection of scramble cells. I, Similarly, MUC16-Cter–overexpressed cell had a lower p53 expression. β-Actin was used as loading control. *, P < 0.05; **, P < 0.01; ***, P < 0.001; and NS, nonsignificant. I, Magnification, X20.

In order to find out the therapeutic role of Muc16 on chemoresistance, we generated a mouse tumor cell line from genetically engineered mouse lung cancer (KrasG12D; AdCre) tissues. The cell line K1418 has endogenous Muc16, and it was stably knocked down by mouse Muc16-specific shRNA (Supplementary Fig. S3D). MTT assays on these cell lines showed that Muc16 knockdown (K1418-shMuc16) cells were more sensitive to cisplatin (Fig. 5E) and gemcitabine (Fig. 5F). These results indicate that MUC16 confers cisplatin and gemcitabine resistance in lung cancer cells.

Mechanism of MUC16-mediated chemoresistance

Upon MUC16 silencing, expression of p53 (wild-type) was increased compared with scramble cells (Fig. 5G). Similarly, the p53 target gene p21 was also upregulated in MUC16 knockdown cells (Supplementary Fig. S3E). We also observed increased expression of p53 in MUC16 knockdown (H292-shMUC16 seq1 and seq2) cells from implanted xenograft tumor tissues (Fig. 5H). Similarly, p53 was downregulated in MUC16-Cter–overexpressed cells (Fig. 5I). Overall, our results indicate that MUC16 regulates TSPYL5 expression, which downregulates p53 and its associated genes, thereby leading to chemoresistance.

Upregulation of MUC16 in cisplatin-resistant lung cancer cells

We developed cisplatin-resistant cell lines by exposing various concentrations (100 nm–3.6 μmol/L) of cisplatin. MUC16 transcript was upregulated in cisplatin-resistant cell lines (P = 0.02) as compared with parental cells (Supplementary Fig. S4A). These results suggest that MUC16 contributes to chemoresistance in lung cancer.

Stable knockdown of TSPYL5 in H292 lung cancer

In order to find out the role of TSPYL5 in lung cancer chemoresistance, we performed stable knockdown of TSPYL5 in H292 cells (Supplementary Fig. S4B and S4C). The p53 expression was increased in TSPYL5 knockdown cells compared with scramble cells (Supplementary Fig. S4B).

Overexpression of MUC16-Cter in MUC16 knockdown H292 cells

To confirm the MUC16-mediated JAK2/STAT3/GR/TSPYL5 signaling in lung cancer, we performed rescue experiments by overexpressing MUC16-Cter in MUC16 knockdown H292 (MUC16-Cter/H292-shMUC16) cells. We observed a restoration of GR and TSPYL5 expression in MUC16-Cter–transfected MUC16 knockdown cells (Fig. 6A). Similarly, p53 expression was downregulated in the presence of MUC16-Cter as compared with vector-transfected MUC16 knockdown (CMV9/H292-shMUC16) cells (Fig. 6A). These results suggest that MUC16 mediates JAK2/STAT3/GR signaling for TSPYL5 gene regulation in lung cancer.

Figure 6.
Figure 6.

Restoration of MUC16 mediated pathways in lung cancer. A, To determine the recue effect of MUC16, we transfected MUC16-Cter (F114HA) in MUC16 knockdown (H292-shMUC16) cells. Restoration of phospho STAT3 (Y705), GR, and TSPYL5 was observed in MUC16-Cter overexpressed in MUC16 knockdown cells as compared with vector-transfected MUC16 knockdown cells. As expected, p53 expression was low in MUC16-Cter overexpressed in MUC16 knockdown cells. Schematic representation for MUC16 signaling in lung cancer cell growth and mechanistic role of MUC16 in chemoresistance in lung cancer. B, MUC16 phosphorylates JAK2 (Y1007/1008) and STAT3 (Y705), leading to translocation of STAT3 into the nucleus, where it recruits the GR. The GR regulates TSPYL5 gene for lung cancer cell growth and metastasis. Inhibition of STAT3 phosphorylation by C188-9 leads to decreased expression of its target gene TSPYL5. In summary, MUC16 promotes JAK2/STAT3/GR signaling axis for TSPYL5 gene expression. This in turn promotes lung cancer cell growth and metastasis. MUC16/TSPYL5 downregulates p53 (wild-type) leading to chemoresistance of lung cancer cells.

Discussion

We observed that MUC16 is overexpressed in human lung adenocarcinoma and in genetically engineered mouse lung cancer tissues (KrasG12D; AdCre), which suggests that MUC16 may have a crucial role in lung cancer pathogenesis.

MUC16 promotes cancer cell growth in breast and pancreas (4, 11, 16). Here, we observed that MUC16 knockdown cells had less growth and tumorigenic properties than control cells. Similarly, MUC16-Cter induced lung cancer cell growth in relative to control cells, suggesting that MUC16 might play a critical role in lung cancer cell growth. In addition, MUC16 was overexpressed in both human primary lung cancer and corresponding lymph node metastases. MUC16 knockdown cells showed significantly reduced migration relative to scramble cells, which suggests that MUC16 may be involved in lung cancer metastasis. Phosphorylation of Src (Y416) was high in MUC16-expressing cells, suggesting that Src phosphorylation is important for MUC16-mediated lung cancer cell migration. Akita and colleagues have reported that the tyrosine phosphorylation of MUC16-Cter is important in ovarian cancer cell migration, and it has been shown that MUC16-Cter interacts with Src family kinases that mediate ovarian cancer cell migration (8). Further, EMT markers were significantly altered based on MUC16 expression. The epithelial marker CK-18 was decreased, and the mesenchymal marker N-cadherin was increased in MUC16-expressing cells where migration was high, thereby suggesting that MUC16 may be involved in the epithelial-to-mesenchymal transition during lung cancer cell metastasis.

Decreased phosphorylation of JAK2 (Y1007/1008) and STAT3 (Y705) was observed in MUC16 knockdown cells. Similarly, increased phosphorylation of JAK2 (Y1007/1008) and STAT3 (Y705) was seen in MUC16-Cter–overexpressed lung cancer, which indicates that MUC16 may mediate JAK2/STAT3 downstream signaling in lung cancer cells. The role of JAK2/STAT3 has been well established in the past, with several studies demonstrating that JAK2/STAT3 signaling is necessary for lung cancer cell growth (33–35).

TSPYL5 has been shown to be involved in cancer cell growth and metastasis in various cancers (17–19). Our microarray data have demonstrated that TSPYL5 was significantly decreased in MUC16 knockdown cells. Further, GR, a regulator for TSPYL5 (by promoter analysis, Biobase software), was also decreased in MUC16 knockdown cells and increased in the MUC16-Cter–overexpressed cells. We have also observed an interaction between STAT3 and GR in lung cancer cells, suggesting that STAT3 binds with GR and regulates TSPYL5 gene expression. Previous studies have demonstrated that the transcription factor STAT3 and GR synergistically regulate various genes (27, 28, 36). Upon STAT3 inhibition, we observed decreased GR and TSPYL5 expression, which suggested that MUC16 regulates GR for TSPYL5 gene expression through its action on STAT3. STAT3 has been shown to recruit the GR and regulate gene expression (20, 27, 28). In support of our findings, the GeneCards database shows that TSPYL5 promoter has GR-binding sites, which suggests that GR regulates TSPYL5 gene expression. Overall, our findings suggest that MUC16 regulates TSPYL5 via JAK2/STAT3/GR signaling axis for lung cancer cell growth and metastasis.

MUC16 has been shown to be involved in chemoresistance of ovarian cancer cells (37); however, the mechanism behind the MUC16-mediated chemoresistance is not well understood. Cisplatin, a platinum analog, is a DNA-damaging agent, widely used in treatment of lung cancer (38, 39). Similarly, gemcitabine is a nucleoside analog that is commonly utilized for the treatment of patients with lung cancer (40). We observed that MUC16 knockdown (both human and mouse tumor) cells were highly sensitive to cisplatin and gemcitabine, whereas MUC16-Cter–overexpressed cells were more resistant. These results suggest that MUC16 might have a role in chemoresistance in lung cancer cells. TSPYL5 has been implicated in radio- and chemoresistance in various cancers including lung and breast cancer (17, 19). Overexpression of TSPYL5 suppresses p53 function and its target genes by regulating USP7 that causes p53 degradation (17). In the present study, TSPYL5 was significantly downregulated in MUC16 knockdown cells. Similarly, expression of p53 and its target gene p21 was increased in MUC16 knockdown cells. In addition, p53 expression was drastically downregulated in MUC16-Cter–overexpressed cells compared with vector cells. Furthermore, increased expression of p53 was observed in MUC16 knockdown cell xenografts, where less tumor growth was seen.

TSPYL5 knockdown in lung cancer cells resulted in an increased expression of p53. These results suggest that MUC16 suppresses p53 via TSPYL5 in lung cancer cells. Furthermore, in cisplatin-resistant lung cancer cells, there was an increased expression of MUC16, which strongly implicates MUC16 in chemoresistance in lung cancer. To confirm the role of MUC16-mediated signaling pathways in lung cancer, we overexpressed MUC16-Cter in MUC16 knockdown down cells and showed the restoration of JAK2/STAT3/GR/TSPYL5 oncogenic signaling pathways. Overall, these results demonstrate that MUC16 regulates TSPYL5, leading to decreased tumor suppressor activity of p53 (41, 42), promoting lung cancer cell growth and chemoresistance.

Conclusion

MUC16 is overexpressed in lung cancer tissues, specifically in adenocarcinoma. MUC16 mediates JAK2/STAT3/GR downstream signaling pathways, resulting in lung cancer cell growth and migration through TSPYL5. In addition, MUC16 confers resistance to cisplatin and gemcitabine by upregulating TSPYL5, which suppresses p53 activity. In conclusion, we found that MUC16 is a key player during lung cancer progression, metastasis, and chemoresistance (Fig. 6B). Targeting MUC16 may increase the response to lung cancer tissues to cytotoxic chemotherapy.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Authors' Contributions

Conception and design: I. Lakshmanan, S.K. Batra, A.K. Ganti

Development of methodology: I. Lakshmanan, S.M. Lele, A.K. Ganti

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): I. Lakshmanan, P. Seshacharyulu, S. Rachagani, A. Thomas, S. Das, P.D. Majhi, R.K. Nimmakayala, R. Vengoji, S.M. Lele, M.P. Ponnusamy, A.K. Ganti

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): I. Lakshmanan, S. Rachagani, S.M. Lele, M.P. Ponnusamy, A.K. Ganti

Writing, review, and/or revision of the manuscript: I. Lakshmanan, P. Seshacharyulu, R.K. Nimmakayala, M.P. Ponnusamy, A.K. Ganti

Study supervision: S.K. Batra, A.K. Ganti

Grant Support

The work is partly supported by grants from the US Department of Veterans' Affairs, UNMC Department of Internal Medicine Summer Undergraduate Research Program, Fred & Pamela Buffett Cancer Center Support Grant (P30CA036727), and NIH (UO1 CA111294, P50 CA127297, U54 CA163120, RO1 CA183459, RO1 CA195586, K22 CA175260, and P20 GM103480).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Acknowledgments

The authors acknowledge the valuable technical support from Kavita Mallya, Microarray Core Facility for gene expression analysis, Cell Sorting Facilities for cell-cycle/apoptosis analysis, and the Confocal Facility for imaging assistance.

Footnotes

  • Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).

  • Received October 11, 2016.
  • Revision received December 12, 2016.
  • Accepted January 28, 2017.

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MUC16 Regulates TSPYL5 for Lung Cancer Cell Growth and Chemoresistance by Suppressing p53
Imayavaramban Lakshmanan, Shereen Salfity, Parthasarathy Seshacharyulu, Satyanarayana Rachagani, Abigail Thomas, Srustidhar Das, Prabin D. Majhi, Rama Krishna Nimmakayala, Raghupathy Vengoji, Subodh M. Lele, Moorthy P. Ponnusamy, Surinder K. Batra and Apar Kishor Ganti
Clin Cancer Res July 15 2017 (23) (14) 3906-3917; DOI: 10.1158/1078-0432.CCR-16-2530
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