A network map of cytoskeleton-associated protein 4 (CKAP4) mediated signaling pathway in cancer

Abstract

Cytoskeleton-associated protein 4 (CKAP4) is a non-glycosylated type II transmembrane protein that serves as a cell surface-activated receptor. It is expressed primarily in the plasma membranes of bladder epithelial cells, type II alveolar pneumocytes, and vascular smooth muscle cells. CKAP4 is involved in various biological activities including cell proliferation, cell migration, keratinocyte differentiation, glycogenesis, fibrosis, thymic development, cardiogenesis, neuronal apoptosis, and cancer. CKAP4 has been described as a pro-tumor molecule that regulates the progression of various cancers, including lung cancer, breast cancer, esophageal squamous cell carcinoma, hepatocellular carcinoma, cervical cancer, oral cancer, bladder cancer, cholangiocarcinoma, pancreatic cancer, myeloma, renal cell carcinoma, melanoma, squamous cell carcinoma, colorectal cancer, and osteosarcoma. CKAP4 and its isoform bind to DKK1 or DKK3 (Dickkopf proteins) or antiproliferative factor (APF) and regulates several downstream signaling cascades. The CKAP4 complex plays a crucial role in regulating the signaling pathways including PI3K/AKT and MAPK1/3. Recently, CKAP4 has been recognized as a potential target for cancer therapy. Due to its biomedical importance, we integrated a network map of CKAP4. The available literature on CKAP4 signaling was manually curated according to the NetPath annotation criteria. The consolidated pathway map comprises 41 activation/inhibition events, 21 catalysis events, 35 molecular associations, 134 gene regulation events, 83 types of protein expression, and six protein translocation events. CKAP4 signaling pathway map data is freely accessible through the WikiPathways Database (https://www.wikipathways.org/index.php/Pathway:WP5322).

Graphical abstract

Generation of CKAP4 signaling pathway map

Supplementary Information

The online version contains supplementary material available at 10.1007/s12079-023-00739-w.

Introduction

Cytoskeleton-associated protein 4 (CKAP4), also known as p63 and cytoskeleton-linking membrane protein (CLIMP-63), identified initially as a shaping protein in the rough endoplasmic reticulum (ER) (Schweizer et al. 1993, 1995). CKAP4 is a non-glycosylated type II transmembrane protein that acts as an activated receptor at the cell surface (Li et al. 2020a, b). The CKAP4 gene is located on human chromosome 12 (12q23.3). In humans, the CKAP4 protein is divided into three regions: an extracellular portion of 475 amino acids (AA 128–602), a single transmembrane region of 22 amino acids (AA 106–127), and an internal domain of 105 amino acids (AA 1–105) (Klopfenstein et al. 2001). CKAP4 is mainly expressed in the plasma membrane of bladder epithelial cells, type II alveolar pneumocytes and vascular smooth muscle cells (Razzaq et al. 2003; Conrads et al. 2006; Gupta et al. 2006). CKAP4 has various ligands such as Dickkopf proteins (Kimura et al. 2016; Kajiwara et al. 2018), antiproliferative factor (APF) (Conrads et al. 2006), surfactant protein A (SP-A) (Gupta et al. 2006), tissue plasminogen activator (tPA) (Razzaq et al. 2003) and alginate exopolysaccharides (Barbier et al. 2012), which determines the different roles induced by the CKAP4 protein.

CKAP4 is involved in various physiological functions, including cell proliferation, cell migration, and stabilizing the structure of the endoplasmic reticulum (ER). It has also been reported that CKAP4 is also implicated as a protumor molecule to promote tumor progression in various cancers including lung, pancreatic, esophageal, and renal tumors (Kimura et al. 2016; Sun et al. 2017; Kajiwara et al. 2018; Shinno et al. 2018). However, reports show that CKAP4 acts as an anticancer protein in various tumors, such as glioma, intrahepatic cholangiocellular carcinoma (ICC), and hepatocellular carcinoma (HCC) (Li et al. 2013, 2014b; Lu et al. 2018). In addition to its role in cancer, CKAP4 has also been implicated in other diseases such as drug-induced cytotoxicity and interstitial cystitis/painful bladder syndrome (IC/PBS) (Conrads et al. 2006; Karasawa et al. 2010). Considering the importance of CKAP4 signaling in various cancers and non-cancerous diseases led us to create a map of the CKAP4 signaling pathway. We generated a signaling pathway map of CKAP4 similar to the previously published pathways related to cancer, including CAMKK2 (Najar et al. 2021), Axl (Dagamajalu et al. 2021), apelin (Dagamajalu et al. 2022a) and DDR1(Dagamajalu et al. 2022b). Using the literature mining, we compiled the downstream molecular events by the interaction between CKAP4 and its ligands into a signaling pathway map. These molecular events have been merged by manual annotation of research articles from the literature, enabling them to be depicted as a single pathway map. The WikiPathways database provides free access to the CKAP4 signaling pathway map.

Methodology

We performed a literature search in PubMed for the research articles relevant to CKAP4-mediated signaling in order to create a CKAP4 signaling pathway map. The research articles were fetched using the following query terms ("CKAP4" OR "cytoskeleton-associated protein 4 " OR "CLIMP-63" OR "cytoskeleton-linking membrane protein 63" OR "p63 " OR “ERGIC-63”) AND (“pathway” OR "signalling" OR "signaling"). The abstract of the research articles was manually screened for CKAP4 signaling-specific molecular events. CKAP4-induced signaling responses were manually curated based on previously published NetPath annotation criteria (Kandasamy et al. 2010). CKAP4 signaling-mediated reactions were grouped into five groups: (i) protein activation/inhibition, (ii) enzyme catalysis/post-translational modifications (PTMs), (iii) protein/gene regulations, (iv) molecular associations, and (v) protein translocation between cell organelles. Information regarding the experiment type, cell lines/tissue used, and details about the sites and residues of PTMs were also curated. PathBuilder, a manual curation software, was used to facilitate the manual curation of signaling events (Kandasamy et al. 2009). The signaling pathway map was visualized by the PathVisio tool (Kutmon et al. 2015).

Results and discussion

The search terms yielded 828 articles pertinent to CKAP4-mediated signaling in PubMed. The 59 articles had specific information on CKAP4-mediated signaling. The manual annotation of these selected articles exposed different events including 41 activation/inhibition, 21 enzyme catalysis, 83 protein expression, 134 gene regulation, 35 molecular association, and 6 translocation events (Supplementary Data S1). These events were merged into a detailed CKAP4 signaling pathway map and made freely accessible through WikiPathways Database (Fig. 1) (https://www.wikipathways.org/index.php/Pathway:WP5322).

Fig. 1.

Schematic representation of CKAP4 mediated signaling pathway, the schematic representation of CKAP4 induced signaling reactions. The signaling pathway map shows molecules associated with ligand-receptor interactions and CKAP4 downstream molecular events, such as enzyme catalysis, protein/gene regulatory events, molecular association, and translocation. The pathway also includes information about the site and residue of post-translational modifications

A summary of the CKAP4 pathway map

CKAP4 activation or overexpression with different ligands leads to multiple downstream signaling pathways under physiological and pathological conditions. For example, DKK1 promotes the formation of a complex between CKAP4 and PIK3R1 (Phosphoinositide-3-kinase regulatory subunit 1) and induces the activation of AKT1 (AKT serine/threonine kinase 1) (Ser 473), leading to increased cell proliferation in normal and cancer cells of pancreatic and lung (Kimura et al. 2016). Upon treatment with SP-A, it binds to its receptor CKAP4, which leads to the translocation of CKAP4 from the endoplasmic reticulum to the cell surface via the activation of PIK3CA and AKT1 (Ser 473) in lung epithelial cells (Kazi et al. 2010). The CKAP4 activation by APF binding inhibits the proliferation of HeLa cervical carcinoma cells (Conrads et al. 2006). The overexpression of CKAP4 in atrial fibroblasts significantly increased protein levels of COL1A1 (Collagen type I alpha 1), FN1 (Fibronectin-1), MMP-1 (Matrix Metalloproteinase-1), TGFB1 (Transforming Growth Factor beta-1), JUN (Jun proto-oncogene), FOS (Fos Proto-Oncogene), and attenuated TIMP-1 (Tissue Inhibitors of Metalloproteinase 1) through the activation of MAPK8/9 (Mitogen-Activated Protein Kinase 8/9) and MAPK14 (Mitogen-Activated Protein Kinase 14), which induces atrial fibrosis and atrial fibrillation (Tan et al. 2021). Recent studies have revealed that CKAP4 plays a significant role in several types of cancer, and it is believed that targeting CKAP4 may offer a potential strategy for cancer treatment.

Breast cancer

In mammary epithelial cells, TGFB1 activates TGFBR1 and induces the activation of FBXO3 (F-Box Protein 3) and regulates the expression of a CKAP4 isoform. This results in reduced expression of TWIST1 (Twist-related protein 1), ZEB1 (Zinc finger E-box-binding homeobox 1) and elevated expression of CDH1 (E-cadherin), DSP (Desmoplakin), PARD3 (Par-3 Family Cell Polarity Regulator). These changes are associated with increased cell migration and invasion and promote tumor metastasis (Niu et al. 2021). A study by Ho et al. (2016), reported that estrogen induces ESR1 activation and translocates to the nucleus, which leads to the upregulation of ΔNp63, and elevated expression of ITGB4, resulting in AKT1 phosphorylation at Ser 473 and increased cell viability and motility in breast cancer cells (Ho et al. 2016). According to Bergholz et al. (2014), the CKAP4 isoform ΔNp63α upregulates the expression of DUSP6 (Dual Specificity Phosphatase 6) in breast cancer cells, which inhibits MAPK1/3 pathway, and attenuated MMP1 and MMP9 expression lead to inhibition of invasion (Bergholz et al. 2014). Buckley et al. (2011), reported that BRCA1 (BReast CAncer gene 1) translocates to the intronic enhancer region of the CKAP4 gene in the nucleus, where it interacts with CKAP4 isoform ΔNp63γ, as well as transcription factor isoforms TFAP2C (AP-2γ) and TFAP2A (AP-2α), leading to the upregulation of transcription of the ΔNp63 isoforms that repress BRCA1, which is one of the important steps in the pathogenesis of basal-like breast cancer (Buckley et al. 2011). A study in breast cancer stem cells reported that CKAP4 directly binds to the regulatory regions of the SHH1 (Sonic Hedgehog 1), GLI2 (GLI Family Zinc Finger 2), and PTCH1 (Protein Patched Homolog 1) genes, leading to an increase in their expression, which eventually increases their capacity of self-renewal (Memmi et al. 2015). In MCF10A breast cancer cells, CKAP4 isoform, ΔNp63α induces the molecular interaction of ΔNp63α and MM1 (Methionine/methionine type 1), which results in downregulation of MM1 and upregulation of MYC (MYC Proto-Oncogene), CDK4 (Cyclin Dependent Kinase 4), and CCND1 (Cyclin D1), which stimulates tumorigenesis and cell cycle progression (Han et al. 2016).

Esophageal squamous cell carcinoma (ESCC)

In esophageal squamous cell carcinoma (ESCC), DKK3 and DKK1 bind to CKAP4 and activate AKT1 phosphorylation at Ser 473, leading to cancer cell proliferation (Kajiwara et al. 2018; Shinno et al. 2018). The overexpression of CKAP4 isoform ΔNp63 leads to phosphorylation of AKT1, which promotes proliferation in BE3 and OE33 human esophageal cancer cell lines through the downregulation of TP53 (Tumor Protein 53) and CDKN1B, and upregulation of CCND1 and CCNE1 (Ye et al. 2014). In the study in ECA109 and EC9706 ESCC cells, the overexpression of CKAP4 isoform, ΔNp63α induces the upregulation of RPS6KA6, which phosphorylates GSK3B at Ser 9 and activates CTNNB1 (Catenin Beta 1) by phosphorylation at Ser 33/Ser 37/Thr 41, which stimulates elevated expression of CD271, ABCG2 (ATP-binding cassette super-family G member 2), BMI-1 (B Lymphoma Mo-MLV Insertion Region 1), NANOG (NK2-family homeobox transcription factor), OCT4 (Octamer-binding transcription factor 4), SOX2 (Sex determining region Y-box 2), MYC, CD44, and TCF1 (T Cell Factor 1), and phosphorylates checkpoint proteins like ATM and CHK2. This enhances radio-resistance and cancer stem-like cell properties (Li et al. 2020a, b). A study by Lee et al. (2014) reported that the overexpression of CKAP4 increased the expression of VIM (Vimentin), TWIST, SUSD2 (Sushi domain containing 2), and PLAU (Plasminogen Activator, Urokinase) through elevated expression of CTNNB1 and upregulation of MYC which are involved in migration and invasion of esophageal squamous carcinoma cells (Lee et al. 2014).

Hepatocellular carcinoma

The overexpression of CKAP4 by APF binding promotes the interaction between CKAP4 and EGFR (Epidermal Growth Factor Receptor) leads to the inhibition of EGFR (by EGF), AKT1 and GSK3B which causes the upregulation of CDH1, TJP1 (Tight Junction Protein-1) and downregulation of VIM, SNAI1 (Snail 1) and prevents the formation of xenograft tumor growth and metastatic potential of hepatocellular carcinoma in nude mice (Li et al. 2014a). Gressner et al. (2005) have found that upon DNA damage, CKAP4 isoform, TAp63α, triggers the upregulation of proapoptotic markers such as CD95, TNF-R1 (Tumor Necrosis Factor Receptor 1), tumor necrosis factor-related apoptosis-inducing ligands (TRAIL-R1, TRAIL-R2), TNF, TRAF (Tumor necrosis factor Receptor–Associated Factor), DAP3 (Death Associated Protein 3), caspases (CASP1, CASP3, CASP4, CASP5, CASP8, CASP9), BCL2L11 (Bcl-2-like protein 11), RAD9 (Cell cycle checkpoint control protein), APAF1 (Apoptotic Protease Activating Factor 1), BAX (Bcl-2-associated X protein) in gene level, and CD95, APAF1, RAD9, CASP1, CASP3, CASP9, BCL2L11, and DAP3 in protein level, which in turn causes apoptosis by activating signaling via death receptors and mitochondria in hepatoma Hep3B cells (Gressner et al. 2005).

Head and Neck cancer

In oral cancer FaDu cells, lovastatin activated PRKAA2, phosphorylated MAPK14, and downregulated the BIRC5 protein. This results in CKAP4 being acetylated and phosphorylated at Ser 160 and Ser 162 to induce apoptosis (Yen et al. 2016). The overexpression of CKAP4 causes the upregulation of PTK2B, FAK, MMP14, JUN at gene and protein levels, activates SRC-PTK2B complex, and phosphorylates SRC at Tyr 416, PTK2B at Tyr 397/576/577, PXN at Tyr 118, AKT1 at Ser 473, JUN at Ser 73, which in turn causes extracellular matrix remodeling, cell migration and invasion in oro-pharyngeal squamous cell cancers (Srivastava et al. 2017). The study by Srivastava et al. (2018), reported that overexpression of CKAP4 isoform, TAp63 prevent the malignant transformation in oral squamous cell carcinoma‑derived cells through the upregulation of NOTCH1 and downregulation of CTNNB1 and LEF1 (Srivastava et al. 2018).

Lung cancer

In non-small-cell lung cancer cells (NSCLC), the overexpression of Golgi phosphoprotein 3 (GOLPH3) interacts with CKAP4 to induce the secretion of exosomes containing CKAP4 and WNT3A (Wnt family member 3A), which induces the upregulation of MYC, TWIST, CCND1, SNAIL, and CD44, resulting in metastasis and the cancer stem cell-like phenotype (Song et al. 2021). The overexpression of β and γ isoforms of CKAP4 induce SHH expression, causing cancer progression in the H1299 non-small lung carcinoma cell line (Caserta et al. 2006).

Cervical cancer

In cervical cancer HeLa cells, APF binding to CKAP4 induces palmitoylation by DHHC2, which is necessary for APF-stimulated translocation of CKAP4 from ER to the plasma membrane and nucleus, which results in an antiproliferative impact by upregulating CDH1 and downregulating VIM, TJP1, and OCLN (Occludin) genes (Planey et al. 2009). Suenaga et al. (2019) reported that activation of CKAP4 isoform, TAp63, causes the downregulation of MYCN, which inhibits tumorigenesis in HeLa cells (Suenaga et al. 2019).

Bladder cancer

APF binding to CKAP4 causes the translocation of CKAP4 to the nucleus where it binds to the promoter of CCN2 (Cellular Communication Network Factor 2) and triggers overexpression of CCN2 protein in T24 bladder carcinoma cells that positively regulates proliferation (Matika et al. 2012). Additionally, overexpression of CKAP4 causes the upregulation of transcription factors including SP1 and E2F1, which downregulates the expression of WASF3 (Wiskott-Aldrich Syndrome protein Family member 3) and enhances invasion after tumorigenesis in bladder cancer by inducing the upregulation of HSPA1A (Heat Shock Protein family A (Hsp70) member 1A) (Jin et al. 2017).

Other cancers

In multiple myeloma cells, DKK1 bind to its receptor CKAP4 induces the molecular association between CKAP4 and CAND1 (Cullin-Associated NEDD8-Dissociated 1) results in the phosphorylation of RELA and its nuclear translocation. The accumulation of RELA in the nucleus leads to the activation of NFKB1A and upregulation of its downstream genes such as IL6, CXCL10, ABCC6, BCL2, which assist in the inducing the drug resistance (Li et al. 2021). The study in ΔNp63α overexpressing MNNG and HOS human osteosarcoma cell lines reported that ΔNp63α promotes the upregulation of miR-527, and miR-665. The miR-527 inhibits TGFBR2, SMAD4 (SMAD Family Member 4), and attenuates TGFBR2, SMAD4 expression, whereas miR-665 inhibits TGFBR2 and phosphorylates SMAD3 which in turn upregulates SERPINE1 expression, and thereby induces metastasis (Rodriguez Calleja et al. 2016). In melanoma cells, CKAP4 upregulation stimulates the phosphorylation of MAP2K1/2, The binding of CKAP4 and MDM2 (Murine Double Minute 2) transports CKAP4 into the cytoplasm where it interacts with FBXW7 (F-box and WD repeat domain containing 7). It induces CKAP4 proteosomal degradation and consequent downregulation of CKAP4 results into decreased cell survival and resistance to MAPK inhibitors in melanoma cells (Patel et al. 2020). The binding of DKK1 to CKAP4 induces the activation of PIK3CA and AKT1, leads to the upregulation of PLVAP (Plasmalemma Vesicle-Associated Protein) and VEGFR2 and increases angiogenesis in cholangiocarcinoma cells (Wang et al. 2021). In pancreatic adenocarcinoma S2-CP8 cells, CKAP4 interacts with ITGB1 and downregulates ITGA5 protein expression, independently of DKK1, which positively regulates cell migration (Osugi et al. 2019). Overexpression of CKAP4 induces anti-apoptosis in renal cell carcinoma cells through the upregulation of CCNB1 (Cyclin B1), CCNB2 (Cyclin B2), and CDK1 proteins (Sun et al. 2017). In human colorectal cancer cells, increased expression of CKAP4 isoform, TAP63 inhibits GSK3B and downregulates the expression of CD74, CD44, PGE2 (Prostaglandin E2), and MIF (Macrophage migration Inhibitory Factor) proteins and thereby suppresses the metastasis (Park et al. 2016). The overexpression of CKAP4 isoform, ΔNp63 induces the upregulation of ANGPTL2 (Angiopoietin-like-2) via NFKB1, which is required for primary tumor-induced neutrophil recruitment in the lung and subsequent pre-metastatic niche formation in mice osteosarcoma cells (Charan et al. 2020). Overexpression of the CKAP4 isoform ΔNp63α causes the attenuation of PTEN (Phosphatase and tensin homolog) via phosphorylation of AKT1, which is involved in increasing keratinocyte proliferation in squamous cell and basal cell carcinomas (Leonard et al. 2011).

Conclusions

The CKAP4 isoforms, such as TAp63 and ΔNp63 are involved in vast array of functions carried out by multiple signaling pathways. CKAP4-mediated signaling is mainly involved in cancer cell proliferation, migration, invasion, differentiation, angiogenesis, metastasis, tumorigenesis, and apoptosis. Understanding the CKAP4 expression level, its functions and downstream molecules of CKAP4 in cancers may provide new insights into CKAP4-based cancer diagnostic and therapeutic approaches for cancer management. The availability of CKAP4-mediated signaling in the public domain will aid biomedicine researchers in understanding the role of various molecules regulating CKAP4 in disease progression. We believe that this resource will help the scientific community to identify new therapeutic drug targets for cancer-related to CKAP4 signaling.

Supplementary Information

Below is the link to the electronic supplementary material.