Integrin αVβ1-activated PYK2 promotes the progression of non-small-cell lung cancer via the STAT3-VGF axis

Abstract

Background: Non-small-cell lung cancer (NSCLC) accounts for 80-85% of all lung cancer and is the leading cause of cancer-related deaths globally. Although various treatment strategies have been introduced, the 5-year survival rate of patients with NSCLC is only 20-30%. Thus, it remains necessary to study the pathogenesis of NSCLC and develop new therapeutic drugs. Notably, PYK2 has been implicated in the progression of many tumors, including NSCLC, but its detailed mechanism remains unclear. In this study, we aimed to elucidate the mechanisms through which PYK2 promotes NSCLC progression.

Methods: The mRNA and protein levels of various molecules were measured using qRT-PCR, western blot (WB), and immunohistochemistry (IHC), respectively. We established stable PYK2 knockdown and overexpression cell lines, and CCK-8, EdU, and clonogenic assays; wound healing, transwell migration, and Matrigel invasion assays; and flow cytometry were employed to assess the phenotypes of tumor cells. Protein interactions were evaluated with co-immunoprecipitation (co-IP), immunofluorescence (IF)-based colocalization, and nucleocytoplasmic separation assays. RNA sequencing was performed to explore the transcriptional regulation mediated by PYK2. Secreted VGF levels were examined using ELISA. Dual-luciferase reporter system was used to detect transcriptional regulation site. PF4618433 (PYK2 inhibitor) and Stattic (STAT3 inhibitor) were used for rescue experiments. A public database was mined to analyze the effect of these molecules on NSCLC prognosis. To investigate the role of PYK2 in vivo, mouse xenograft models of lung carcinoma were established and examined.

Results: The protein level of PYK2 was higher in human NSCLC tumors than in the adjacent normal tissue, and higher PYK2 expression was associated with poorer prognosis. PYK2 knockdown inhibited the proliferation and motility of tumor cells and caused G1-S arrest and cyclinD1 downregulation in A549 and H460 cells. Meanwhile, PYK2 overexpression had the opposite effect in H1299 cells. The siRNA-induced inhibition of integrins alpha V and beta 1 led to the downregulation of p-PYK2(Tyr402). Activated PYK2 could bind to STAT3 and enhance its phosphorylation at Tyr705, regulating the nuclear accumulation of p-STAT3(Tyr705). This further promoted the expression of VGF, as confirmed by RNA sequencing in a PYK2-overexpressing H1299 cell line and validated by rescue experiments. Two sites in promoter region of VGF gene were confirmed as binding sites of STAT3 by Dual-luciferase assay. Data from the TGCA database showed that VGF was related to the poor prognosis of NSCLC. IHC revealed higher p-PYK2(Tyr402) and VGF expression in lung tumors than in adjacent normal tissues. Moreover, both proteins showed higher levels in advanced TNM stages than earlier ones. A positive linear correlation existed between the IHC score of p-PYK2(Tyr402) and VGF. Knockdown of VGF inhibited tumor progression and reversed the tumor promoting effect of PYK2 overexpression in NSCLC cells. Finally, the mouse model exhibited enhanced tumor growth when PYK2 was overexpressed, while the inhibitors PF4618433 and Stattic could attenuate this effect.

Conclusions: The Integrin αVβ1-PYK2-STAT3-VGF axis promotes NSCLC development, and the PYK2 inhibitor PF4618433 and STAT3 inhibitor Stattic can reverse the pro-tumorigenic effect of high PYK2 expression in mouse models. Our findings provide insights into NSCLC progression and could guide potential therapeutic strategies against NSCLC with high PYK2 expression levels.

Keywords: Integrin αVβ1; Non-small-cell lung cancer (NSCLC); Nuclear accumulation of p-STAT3(Tyr705); PYK2; VGF.

Fig. 1

PYK2 and p-PYK2(Tyr402) are highly expressed in NSCLC tissues and PTK2B mRNA is associated with a poor prognosis. A PYK2 protein expression level of NSCLC tissues is higher than paired normal lung tissues. B IHC indicate the higher expression of p-PYK2(Tyr402) in NSCLC tumor than adjacent nontumor tissue, and higher expression in later TNM stage than earlier TNM stage. Scale bar, 100 μm. CPTK2B expression level is negatively related with the overall survival of NSCLC patients. The expression and prognostic data were downloaded from the TCGA database. D-EPTK2B mRNA and PYK2, p-PYK2(Tyr402) protein levels in NSCLC cell lines. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001

Fig. 2

Inhibition of NSCLC cell proliferation and cell cycle G1-S arrest by knockdown of PYK2. A-B PYK2 mRNA and protein levels were knockdown in A549 and H460 sh-PYK2 cells. C-D CCK-8 assay was used to detect cell viability in A549 and H460 cells; cell viability was determined at 24, 48, and 72 h. PYK2-knockdown inhibited growth of A549 and H460 cells. E PYK2-knockdown inhibited the clonogenic ability of A549 and H460 cells. F-G EdU further confirmed that PYK2 knockdown inhibited cell proliferation. Scale bar, 200 μm. H-I Knockdown of PYK2 caused cell cycle G1-S arrest of A549 and H460 cells. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001

Fig. 3

PYK2-overexpression promoted the proliferation of H1299 cells, knockdown of PYK2 attenuates tumor growth in a murine xenograft model. A-B PYK2 mRNA and protein levels increased in H1299 PYK2 overexpressed cells. C CCK-8 assay to detect cell viability in H1299 cells; PYK2-overexpression promoted the clonogenic ability of H1299 cells. D EdU further confirmed that PYK2 overexpression enhanced cell proliferation. Scale bar, 200 μm. E Overexpression of PYK2 caused cell cycle G1 stage transfer to S stage in H1299 cells. F PYK2-knockdown in A549 cells xenografts in nude mice (n = 6) at the experimental endpoint. Tumors were dissected and photographed as shown. G Tumor growth curves in mice (n = 6 in each group). H Each tumor formed was weighted. I-J WB and IHC detected p-PYK2 expression and the total PYK2 expression in tumor cell. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001

Fig. 4

Knockdown of Integrin αVβ1 could inhibit the tyrosine 402 site phosphorylation of PYK2. A Transient transfection of siRNAs of Integrin α3, α5, αV, α6, and β1 to screen which one could affect the activation of PYK2 and WB to detect p-PYK2(tyr402) in knockdown of each Integrin in A549 cells; B CO-IP demonstrated the interaction of PYK2 and Integrin αV, β1.C-D Colocalization of PYK2 and Integrin αV, β1 (PYK2, SANTACRUZ, sc-393,181, Mouse for A and PYK2, abcam, ab32571, Rabbit for B) by IF. Scale bar, 20 μm

Fig. 5

p-PYK2(Tyr402) interacts with STAT3 and can regulate the tyrosine 705 phosphorylation of STAT3 and p-STAT3(Tyr705) nuclear accumulation. A GSEA analysis showed a positive relation between PYK2 and the JAK-STAT signaling pathway. B Level of total STAT3 and p-STAT3(Tyr705) in A549, H460 stable PYK2 knockdown stable cell lines and H1299 stable PYK2 overexpressed cell line. C p-PYK2(Tyr402) and STAT3 colocalized in A549 and H460 cells. D Coimmunoprecipitation assay of exogenously expressed PYK2 and STAT3 plasmid in HEK293T cell demonstrated the interaction of these two proteins. E p-PYK2(Tyr402) and STAT3 could be directly bound to each other. F-G Nucleocytoplasmic separation and immunofluorescence experiments showed more p-STAT3(Tyr705) nuclear accumulation in PYK2 overexpressed compared to the vector

Fig. 6

PYK2 regulates VGF expression through p-STAT3(Tyr705). A VGF in volcano plot of RNA sequencing based on H1299 PYK2 overexpressed cells compared to vector. B VGF mRNA reduced in two stable knockdown cell lines. C IHC of VGF; its scores were higher in tumor than paired adjacent normal lung tissue; higher in advanced TNM stage than earlier stage; IHC scores of p-PYK2(Tyr402) and VGF have a linear correlation. Scale bar, 100 μm. D Serum VGF of healthy control was lower than that in NSCLC patients. E Negative effect of VGF expression on overall survival prognosis of NSCLC patients from TCGA database. F Treatment with the p-PYK2(Tyr402) inhibitor PF4618433 20µMand p-STAT3(Tyr705) inhibitor Stattic 5µM reversed the increase in VGF mRNA. G The luciferase assay confirmed STAT3 transcriptionally regulated VGF. H Protein of VGF in stable knockdown cells and H1299 PYK2 OE cell lines with PF4618433 20µM and Stattic 5µM treated or not. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001

Fig. 7

PYK2 inhibitor PF4618433 and STAT3 inhibitor Stattic reversed the enhancing effect of PYK2-overexpressing for xenograft subcutaneous tumor growth in vivo. A-B The rescue phenotypic experiments with Stattic 5µM attenuated the pro-cancer effect of PYK2 overexpression in CCK-8 and colony formation. C Flow chart of this animal experiment. D-F Tumor photos and growth curves, tumor weight at execution of vector, PYK2 OE, PYK2 OE + PF4618433, PYK2 OE + Stattic. G WB of each group for PYK2, p-PYK2(Tyr402), STAT3, p-STAT3(Tyr705) and VGF. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001

Fig. 8

Pathway figure of this study