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’s 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 to normal. MUC16 knockdown (KD) in various 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), while 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 over-expression 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 down regulation 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 JAK2/STAT3/GR axis.
<p>Pharmacological inhibition of phospho JAK1/2 in lung cancer cells. A, Upon JAK1/2 inhibition by ruxolitinib, we observed decreased phosphorylation of STAT3 (Y705) but no change in STAT3 expression. B, Similar effect was also observed in MUC16-Cter overexpressed lung cancer. β-actin was used as loading control.</p>
<p>Pharmacological inhibition of phospho JAK1/2 in lung cancer cells. A, Upon JAK1/2 inhibition by ruxolitinib, we observed decreased phosphorylation of STAT3 (Y705) but no change in STAT3 expression. B, Similar effect was also observed in MUC16-Cter overexpressed lung cancer. β-actin was used as loading control.</p>
<p>MUC16 incidence in lung cancer. A, MUC16 is overexpressed in a greater proportion of lung adenocarcinoma compared to other histological subtypes. B, Data from TCGA also shows that MUC16 is highly expressed in adenocarcinoma tissues than squamous carcinoma tissues. C, TSPYL5 transcript was significantly decreased in MUC16 knockdown (H292-shMUC16 seq1 and shMUC16 seq2) as validated by quantitative real-time PCR analysis. D, Immunoprecipitation assay demonstrated that STAT3 interacts with GR in lung cancer cells. **P<0.01.</p>
<p>Chemoresistance properties of MUC16 and effect of cisplatin on apoptosis of MUC16 knockdown cells. A & B, MUC16 knockdown (H1975-shMUC16 seq1 and 2) cells were highly sensitive to cisplatin (A) and gemcitabine (B). C, The percentage of apoptotic cells was significantly higher in MUC16 knockdown cells (H292-shMUC16) treated with 5μM cisplatin. In contrast, no significant change was observed in the untreated scramble (H292-SCR) and MUC16 knockdown cells. D, We performed stable knockdown of Muc16 in K1418, the result shows that Muc16 is significantly decreased as compared to scramble cells. E, The p53 target gene p21 was significantly increased in MUC16 knockdown (H292-shMUC16) cells. *P<0.05, **P<0.01, ***P<0.001, and NS non-significant.</p>
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