Multitranscriptome analyses of keloid fibroblasts reveal the role of the HIF-1α/HOXC6/ERK axis in keloid development

Abstract

Background: A keloid is a disease of excessive fibrosis that is characterized by the aberrant proliferation of fibroblasts. However, the molecular mechanisms of fibroblasts during the development of keloids remain unclear. This study aims to identify new molecular targets that promote the proliferation and migration of keloid fibroblasts, providing new ideas for the prevention and treatment of keloids. Methods: We utilized bioinformatics tools to analyze data from keloid fibroblasts (KFs) available in the Gene Expression Omnibus (GEO) database to identify the key genes involved in keloid development. Homeobox C6 (HOXC6) emerged as a hub gene in KFs from the GEO database was verified in keloid tissue samples and KFs using reverse transcription-quantitative polymerase chain reaction, western blot (WB) and immunohistochemistry. Subsequently, the effects of down-regulated HOXC6 expression on the cellular behaviors of KFs were examined by performing Cell Counting Kit-8, flow cytometry, transwell migration and WB assays. Meanwhile, we performed transcriptome sequencing and gene set enrichment analysis (GSEA) to further explore HOXC6-related mechanisms and validated the signaling pathways by performing a series of experiments. Results: HOXC6 was the top-ranking hub gene of KFs in microarray datasets from GEO and was upregulated in keloid tissue samples and KFs. Downregulation of HOXC6 inhibited proliferation, migration and extracellular matrix (ECM) accumulation and promoted KF apoptosis. GSEA predicted that the hypoxia signaling pathway was associated with HOXC6 in KFs. Transcriptome sequencing suggested that the extracellular regulated protein kinase (ERK) pathway was one of the downstream pathways of HOXC6 in KFs. Our experiments confirmed that hypoxia-inducible factor-1α (HIF-1α) upregulates HOXC6, contributing to KFs proliferation, migration, apoptosis inhibition and collagen accumulation through the ERK signaling pathway. Conclusions: Our findings first revealed that HOXC6 acts as an oncogenic driver in the molecular mechanisms of fibroblasts in keloids. The HIF-1α/HOXC6/ERK axis promotes proliferation, migration and ECM production by KFs, contributing to the progression of keloids. Taken together, HOXC6 may serve as a promising novel therapeutic target and new focus for research designed to understand the pathogenesis of keloids.

Keywords

Keloid / Fibroblast / HOXC6 / HIF-1α / ERK / Transcriptome

References

[1] Berman B, Maderal A, Raphael B. Keloids and hypertrophic scars: pathophysiology, classification, and treatment. Dermatol Surg. 2017; 43: S3-18.

[2] Satish L, Evdokiou A, Geletu E, Hahn JM, Supp DM. Pirfenidone inhibits epithelial-mesenchymal transition in keloid keratinocytes. Burns Trauma. 2020; 8: tkz007. https://doi.org/10.1093/burnst/tkz007.

[3] Limandjaja GC, Niessen FB, Scheper RJ, Gibbs S. The keloid disorder: heterogeneity, histopathology, mechanisms and models. Front Cell Dev Biol. 2020; 8: 360.

[4] Akiyama M.Pustular psoriasis as an autoinflammatory keratinization disease (AiKD): genetic predisposing factors and promising therapeutic targets. J Dermatol Sci. 2022; 105: 11-7.

[5] Deng C-C, Hu Y-F, Zhu D-H, Cheng Q, Gu J-J, Feng Q-L, et al. Single-cell RNA-seq reveals fibroblast heterogeneity and increased mesenchymal fibroblasts in human fibrotic skin diseases. Nat Commun. 2021; 12: 3709.

[6] Jung J, Jeong S, Jeong H, Oh HE, Choi J-W, Lee ES, et al. Increased HOXC6 mRNA expression is a novel biomarker of gastric cancer. De Re V, editor PLOS ONE. 2020; 15: e0236811. https://doi.org/10.1371/journal.pone.0236811.

[7] Stelnicki EJ, Kömüves LG, Kwong AO, Holmes D, Klein P, Rozenfeld S, et al. HOX Homeobox genes exhibit spatial and temporal changes in expression during human skin development. J Invest Dermatol. 1998; 110: 110-5.

[8] Scott GA, Goldsmith LA. Homeobox genes and skin development: a review. J Invest Dermatol. 1993; 101: 3-8.

[9] Xie Q, Liu D, Yu M, Wu X, Zhao Y, Hu Q, et al. Homeobox B9 facilitates hypertrophic scar formation via activating the mitogen-activated protein kinase signaling pathway. Mol Med Rep. 2017; 16: 1669-76.

[10] Aberdam D, Negreanu V, Sachs L, Blatt C. The oncogenic potential of an activated Hox-2.4 homeobox gene in mouse fibroblasts. Mol Cell Biol. 1991; 11: 554-7.

[11] Maulbecker CC, Gruss P. The oncogenic potential of deregulated homeobox genes. Cell Growth Differ Mol Biol J Am Assoc Cancer Res. 1993; 4: 431-41.

[12] Kanzler B, Viallet JP, Mouellic HL, Boncinelli E, Duboule D, Dhouailly D. Differential expression of two different homeobox gene families during mouse tegument morphogenesis. Int J Dev Biol. 1994; 38: 633-40.

[13] Moon S-M, Kim S-A, Yoon J-H, Ahn S-G. HOXC6 is deregulated in human head and neck squamous cell carcinoma and modulates Bcl-2 expression. J Biol Chem. 2012; 287: 35678-88.

[14] Bodey B, Siegel SE, Kaiser HE. Immunocytochemical detection of the homeobox B3, B4, and C6 gene products in breast carcinomas. Anticancer Res. 2000; 20: 3281-6.

[15] Fujiki K, Duerr E, Kikuchi H, Ng A, Xavier RJ, Mizukami Y, et al. Hoxc6 is overexpressed in gastrointestinal carcinoids and interacts with JunD to regulate tumor growth. Gastroenterology. 2008; 135: 907-16.e2.

[16] Bodey B, Siegel SE, Luck JV, Kaiser HE. Homeobox B3, B4, and C6 gene product expression in osteosarcomas as detected by immunocytochemistry. Anticancer Res. 2000; 20: 2717-21.

[17] Kischer CW, Thies AC, Chvapil M. Perivascular myofibroblasts and microvascular occlusion in hypertrophic scars and keloids. Hum Pathol United States. 1982; 13: 819-24.

[18] Zhao B, Guan H, Liu J-Q, Zheng Z, Zhou Q, Zhang J, et al. Hypoxia drives the transition of human dermal fibroblasts to a myofibroblast-like phenotype via the TGF-β1/Smad3 pathway. Int J Mol Med. 2017; 39: 153-9.

[19] Wulandari E, Jusman SWA, Moenadjat Y, Jusuf AA, Sadikin M. Expressions of collagen I and III in hypoxic keloid tissue. Kobe J Med Sci. 2016; 62: E58-69.

[20] Ma X, Chen J, Xu B, Long X, Qin H, Zhao RC, et al. Keloid-derived keratinocytes acquire a fibroblast-like appearance and an enhanced invasive capacity in a hypoxic microenvironment in vitro. Int J Mol Med. 2015; 35: 1246-56.

[21] Kang Y, Roh MR, Rajadurai S, Rajadurai A, Kumar R, Njauw C-N, et al. Hypoxia and HIF-1α regulate collagen production in keloids. J Invest Dermatol. 2020; 140: 2157-65.

[22] Lei R, Li J, Liu F, Li W, Zhang S, Wang Y, et al. HIF-1α promotes the keloid development through the activation of TGF-β/Smad and TLR4/MyD88/NF-κB pathways. Cell Cycle. 2019; 18: 3239-50.

[23] Hahn JM, Glaser K, Mcfarland KL, Aronow BJ, Boyce ST, Supp DM. Keloid-derived keratinocytes exhibit an abnormal gene expression profile consistent with a distinct causal role in keloid pathology. Wound Repair Regen. 2013; 21: 530-44.

[24] Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, et al. Limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015; 43:gkv007. https://doi.org/10.1093/nar/gkv007.

[25] Chen C, Chen H, Zhang Y, Thomas HR, Frank MH, He Y, et al. TBtools: an integrative toolkit developed for interactive analyses of big biological data. Mol Plant. 2020; 13: 1194-202.

[26] Yu G, Wang LG, Han Y, He QY. clusterProfiler: an R package for comparing biological themes among gene clusters. Omics-J Integr Biol. 2012; 16: 284-7.

[27] Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005; 102: 15545-50.

[28] Franceschini A, Szklarczyk D, Frankild S, Kuhn M, Simonovic M, Roth A, et al. STRING v9.1: protein-protein interaction networks, with increased coverage and integration. Nucleic Acids Res. 2012; 41: D808-15.

[29] Smoot ME, Ono K, Ruscheinski J, Wang P-L, Ideker T. Cytoscape 2.8: new features for data integration and network visualization. Bioinformatics. 2011; 27: 431-2.

[30] Chin C-H, Chen S-H, Wu H-H, Ho C-W, Ko M-T, Lin C-Y. cytoHubba: identifying hub objects and sub-networks from complex interactome. BMC Syst Biol. 2014; 8: S11. https://doi.org/10.1186/1752-0509-8-S4-S11.

[31] Farag AGA, Samaka R, Elshafey EN, Shehata WA, El Sherbiny EG, Hammam MA. Immunohistochemical study of janus kinase 1/signal transducer and activator of transcription 3 in psoriasis vulgaris. Clin Cosmet Investig Dermatol. 2019; 12: 497-508.

[32] Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014; 15: 550. https://doi.org/10.1186/s13059-014-0550-8.

[33] Wu T, Hu E, Xu S, Chen M, Guo P, Dai Z, et al. clusterProfiler 4.0: a universal enrichment tool for interpreting omics data. The. Innovation (N Y). 2021; 2: 100141. https://doi.org/10.1016/j.xinn.2021.100141.

[34] Krumlauf R.Hox genes, clusters and collinearity. Int J Dev Biol. 2018; 62: 659-63.

[35] Chang S-L, Chan T-C, Chen T-J, Lee S-W, Lin L-C, Win KT. HOXC6 overexpression is associated with Ki-67 expression and poor survival in NPC patients. J Cancer. 2017; 8: 1647-54.

[36] Tucci R, Campos MS, Matizonkas-Antonio LF, Durazzo M, Pinto Junior D d S, Nunes FD. HOXB5 expression in oral squamous cell carcinoma. J Appl Oral Sci. 2011; 19: 125-9.

[37] Zhang F, Ren C-C, Liu L, Chen Y-N, Yang L, Zhang X-A. HOXC6 gene silencing inhibits epithelial-mesenchymal transition and cell viability through the TGF-β/smad signaling pathway in cervical carcinoma cells. Cancer Cell Int. 2018; 18: 204. https://doi.org/10.1186/s12935-018-0680-2.

[38] Hahn JM, Mcfarland KL, Combs KA, Anness MC, Supp DM. Analysis of HOX gene expression and the effects of HOXA9 overexpression in fibroblasts derived from keloid lesions and normal skin. Wound Repair Regen. 2021; 29: 777-91.

[39] Jin J, Zhai H-F, Jia Z-H, Luo X-H. Long non-coding RNA HOXA11-AS induces type I collagen synthesis to stimulate keloid formation via sponging miR-124-3p and activation of Smad5 signaling. Am J Physiol-Cell Physiol. 2019; 317: C1001-10.

[40] Ji M, Feng Q, He G, Yang L, Tang W, Lao X, et al. Silencing homeobox C6 inhibits colorectal cancer cell proliferation. Oncotarget. 2016; 7: 29216-27.

[41] Li P-D, Chen P, Peng X, Ma C, Zhang W-J, Dai X-F. HOXC6 predicts invasion and poor survival in hepatocellular carcinoma by driving epithelial-mesenchymal transition. Aging. 2018; 10: 115-30.

[42] Albadari N, Deng S, Li W. The transcriptional factors HIF-1 and HIF-2 and their novel inhibitors in cancer therapy. Expert Opin Drug Discovery. 2019; 14: 1-16.

[43] Cho J-W, Il K-J, Lee K-S. Downregulation of type I collagen expression in silibinin-treated human skin fibroblasts by blocking the activation of Smad2/3-dependent signaling pathways: potential therapeutic use in the chemoprevention of keloids. Int J Mol Med. 2013; 31: 1148-52.

[44] Jusman S, Sari DH, Ningsih SS, Hardiany NS, Sadikin M. Role of hypoxia inducible Factor-1 alpha (HIF-1α) in Cytoglobin expression and fibroblast proliferation of keloids. Kobe J Med Sci. 2019; 65: E10-8.

[45] Seifert O, Bayat A, Geffers R, Dienus K, Buer J, Löfgren S, et al. Identification of unique gene expression patterns within different lesional sites of keloids. Wound Repair Regen. 2008; 16: 254-65.

[46] Shah N, Sukumar S. The Hox genes and their roles in oncogenesis. Nat Rev Cancer. 2010; 10: 361-71.

[47] Moon Y, Moon R, Roh H, Chang S, Lee S, Park H. HIF-1α-dependent induction of carboxypeptidase A4 and carboxypeptidase E in hypoxic human adipose-derived stem cells. Mol Cells. 2020; 43: 945-52.

[48] Guo YJ, Pan WW, Liu SB, Shen ZF, Xu Y, Hu LL. ERK/MAPK signalling pathway and tumorigenesis (review). Exp Ther Med. 2020; 19: 1997-2007.

[49] Huang W, Hua H, Xiao G, Yang X, Yang Q, Jin L. ZC3HAV1 promotes the proliferation and metastasis via regulating KRAS in pancreatic cancer. Aging. 2021; 13: 18482-97.

[50] Zhang X, Xie J, Sun H, Wei Q, Nong G. miR-29a-3p regulates the epithelial-mesenchymal transition via the SPARC/ERK signaling pathway in human bronchial epithelial cells. Int J Mol Med. 2021; 48: 171. https://doi.org/10.3892/ijmm.2021.5004.

[51] Yang P, Kang W, Pan Y, Zhao X, Duan L. Overexpression of HOXC6 promotes cell proliferation and migration via MAPK signaling and predicts a poor prognosis in glioblastoma. Cancer Manag Res. 2019; 11: 8167-79.

[52] Satish L, Babu M, Tran KT, Hebda PA, Wells A. Keloid fibroblast responsiveness to epidermal growth factor and activation of downstream intracellular signaling pathways. Wound Repair Regen. 2004; 12: 183-92.