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Hedgehog ligand and receptor cooperatively regulate EGFR stability and activity in non-small cell lung cancer

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Abstract

Purpose

The hyperactivation of epidermal growth factor receptor (EGFR) plays a crucial role in non-small cell lung cancer (NSCLC). Hedgehog (Hh) signaling has been implicated in the tumorigenesis and progression of various cancers, however, its function in NSCLC cells remains controversial. Herein, we present a novel finding that challenges the current understanding of Hh signaling in tumor growth.

Methods

Expression of Hh ligands and receptor were assessed using TCGA datasets, immunoblotting and immunohistochemical. Biological function of Hh ligands and receptor in NSCLC were tested using colony formation, cell count kit-8 (CCK-8) and xenograft assays. Biochemical effect of Hh ligands and receptor on regulating EGFR stability and activity were checked via immunoblotting.

Results

Expression of Hh ligands and receptor was suppressed in NSCLC tissues, and the lower expression levels of these genes were associated with poor prognosis. Ptch1 binds to EGFR and facilitates its poly-ubiquitylation and degradation independent of downstream transcriptional signaling. Moreover, Hh ligands cooperate with Ptch1 to regulate the protein stability and activity of EGFR. This unique mechanism leads to a suppressive effect on NSCLC tumor growth.

Conclusion

Non-canonical Hh signaling pathway, involving cooperation between Hh ligands and their receptor Ptch1, facilitates the degradation of EGFR and attenuates its activity in NSCLC. These findings provide novel insights into the regulation of EGFR protein stability and activity, offer new diagnostic indicators for molecular typing of NSCLC and identify potential targets for targeted therapy of this challenging disease.

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Data availability

Not applicable.

Abbreviations

BCC:

Basal cell carcinoma

CCK-8:

Cell count kit-8

CHX:

Cycloheximide

Co-IP:

Co-immunoprecipitation

Dhh:

Desert Hedgehog

EGFR:

Epidermal growth factor receptor

EMT:

Epithelial-mesenchymal transition

Her2:

Human epidermal growth factor receptor 2

Hh:

Hedgehog

ICD:

Intracellular domain

IHC:

Immunohistochemical

Ihh:

Indian Hedgehog

NSCLC:

Non-small cell lung cancer

Ptch1:

Patched-1

Shh:

Sonic Hedgehog

TCGA:

The Cancer Genome Atlas

TKIs:

Tyrosine kinase inhibitors

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Funding

This work was supported in part by the grants from the National Natural Science Foundation of China (No. 32260160 to M.C., No. 82160507 to H.W. and 32070783 to S.L.), and the Natural Science Foundation of Jiangxi Province (No. 20212ACB206009 to M.C., 20202BBG72003 to S.L., and 20232ACB206034 to H.W.).

Author information

Author notes

  1. Aidi Huang, Junyao Cheng and Yuan Zhan contributed equally to this work.

Authors and Affiliations

  1. Jiangxi Clinical Research Center for Respiratory Diseases, Jiangxi Institute of Respiratory Disease, The Department of Respiratory and Critical Care Medicine, the First Affiliated Hospital, Jiangxi Medical College, Nanchang University, 330006, Nanchang, Jiangxi, China

    Aidi Huang, Junyao Cheng, Yanlu Xuan, Xinping Xu & Minzhang Cheng

  2. Jiangxi Key Laboratory of Molecular Diagnostics and Precision Medicine, Center for Experimental Medicine, the First Affiliated Hospital, Jiangxi Medical College, Nanchang University, 330006, Nanchang, Jiangxi, China

    Aidi Huang, Junyao Cheng, Feifei Zhou, Yanlu Xuan, Hailong Wang, Shiwen Luo & Minzhang Cheng

  3. Institute of Molecular Pathology, Department of Pathology, the First Affiliated Hospital, Jiangxi Medical College, Nanchang University, 330006, Nanchang, Jiangxi, China

    Yuan Zhan

  4. Department of Gastroenterology, Shandong Provincial Hospital Affiliated to Shandong First Medical University, 250021, Jinan, Shandong, China

    Feifei Zhou

  5. Department of Oncology, the First Affiliated Hospital, Jiangxi Medical College, Nanchang University, 330006, Nanchang, Jiangxi, China

    Yiting Wang

  6. Department of Nuclear Medicine, the First Affiliated Hospital, Jiangxi Medical College, Nanchang University, 330006, Nanchang, Jiangxi, China

    Qingjie Chen

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Contributions

M.C. and S.L. designed all the experiments; A.H., J.C., M.C., and H.W. conducted the function assays in cell lines and nude mice; Y.Z., Y.W., Q.C., and X.X. conducted the IHC staining and clinical investigations; A.H., J.C., Y.X. and F.Z. handled all the biochemical analysis; M.C. and S.L. analyzed and organized the data; A.H., M.C. and S.L. wrote the manuscript; and all the authors revised the manuscript.

Corresponding authors

Correspondence to Shiwen Luo or Minzhang Cheng.

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Ethics approval and consent to participate

All experiments with human tissue samples were approved and supervised by the Ethics Committee of the First Affiliated Hospital of Nanchang University ([2022]CDYFYYLK[06–023]). Protocols for animal experiments were approved by the Ethical Committee of the First Affiliated Hospital of Nanchang University and conformed to the guidelines of the National Institutes of Health on the Ethical Use of Animals.

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The authors declare no competing interests.

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Supplementary Material 1

: Supplementary Fig. 1. Supplementary to Fig. 1. A. Immunohistochemical assays confirmed that the protein levels of EGFR and p-EGFR were higher in lung adenocarcinoma tissues than in normal lung tissue. n = 5, Scale bars, 100 μm (above), 50 μm (below)

Supplementary Material 2

: Supplementary Fig. 2. Supplementary to Fig. 4. A. Construction of an overexpression Ptch1 cell line in PC-9 cells. B. Ptch1 decreases the protein level of EGFR in PC-9 cells. C. Representative images showing IHC staining of both human control and Ptch1 tissues in the section stained for EGFR, Ptch1 and Ki67 detection. Scale bars, 100 μm (200x), 50 μm (400x)

Supplementary Material 3

: Supplementary Fig. 3. Supplementary to Fig. 6. A. Ptch1 regulates EGFR independent of the transcriptional activity of targets downstream of the canonical Hh signaling. HEK-293T cells were transfected with Flag-Ptch1 and cultured for 48 h. They were then pretreated with N-Shh conditional medium with or without the Gli inhibitor GANT61 before harvesting. The lysates were examined via IB with the indicated antibodies. B. mRNA levels of Hh target genes in PC-9 cells with or without stable Ptch1 expression and treated with GFP or N-Shh was determined by qPCR. C-D. HEK-293T cells transfected with Flag-Ptch1 were treated with N-Shh conditional medium in a time gradient, and Hh ligand stimulation was shown to induce Ptch1-mediated EGFR degradation. E. In A549 cells, the expression of Ptch1 did not affect the protein level of EGFR. F-G. A549 cells stably expressing Lv-Ptch1 were treated with N-Shh conditional medium in a time gradient, and Hh ligand stimulation was shown to promote EGFR degradation after Ptch1 is expressed. H-I. Time-gradient EGF stimulation activates EGFR phosphorylation, and the effect of the Hh ligands and receptor Ptch1 on EGFR phosphorylation was measured. Hh ligands synergize with Ptch1 to inhibit EGFR activity. The data are presented as the mean ± SD (n = 3), ***p < 0.001, n.s., not significance. J. Immunofluorescence for co-localization of EGFR with EEA1 in overexpressing Ptch1 and control cells treated with GFP or N-shh conditioned medium. Scale bars, 10 μm. K. There was no significant effect on the co-localization of EGFR and EEA1 in overexpressing Ptch1 and control cells treated with GFP or N-shh conditioned medium, The data are presented as the mean ± SD (n = 3), ***p < 0.001, n.s., not significance

Supplementary Material 4

: Supplementary table 1 Primers

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Huang, A., Cheng, J., Zhan, Y. et al. Hedgehog ligand and receptor cooperatively regulate EGFR stability and activity in non-small cell lung cancer. Cell Oncol. (2024). https://doi.org/10.1007/s13402-024-00938-6

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