Xu Chen,Hongling Liu,Fenghui Zhao＊,Zongxian Jiao,Jinsui Wang,Yamei Dang

1Department of Pathology,Gansu Provincial Hospital,Lanzhou 730000,China

2Department of Occupational Medicine,the Third Gansu Provincial Hospital,Lanzhou 730000,China

3Pathological Institution of Basic Medical College,Lanzhou University,Lanzhou 730000,China

Key words:Wnt5a; cancer; methyl modification

Abstract Wnt5a is a representative Wnt ligand that regulates multiple cellular functions through the Wnt5a nonclassical pathway.Although Wnt5a has been implicated in various pathological conditions,its role in cancer is ambiguous and might involve methyl modifications,distinct mRNA isoforms,as well as different downstream pathways.Therefore,it is an essential factor in cancers’ progression (invasion,migration,proliferation,and epithelial-mesenchymal transition),and a potential biomarker for prognosis and treatment.

WNT factors regulate various cellular functions,including proliferation,migration and differentiation.[1,2]They encompass over 19 members in mice and humans,and are modified by cysteine-rich secretory ligands.[3]Based on the downstream signaling pathways,Wnt factors are classified into the β-catenin-dependent(e.g.,Wnt1,Wnt3,Wnt3a and Wnt7a) and β-catenin-independent (e.g.,Wnt2,Wnt4,Wnt5a,Wnt5b,Wnt6,Wnt7b and Wnt11) pathway activators.[4]Wnt5a is highly expressed during embryonic development,and declines thereafter in normal adult tissues.[5,6]Aberrant Wnt5a expression levels have been reported in various cancers,wherein it plays contrasting roles.[7,8]For example,its short isoform (Wnt5a-S) containing 319 amino acids promotes cancer cell proliferation,while its longer isoform (Wnt5a-L) with 337 amino acids has an inhibitory effect and it is regulated by hypermethylation of WNT5A promotor region.[9]In this review,the role of Wnt5a in cancers and the associated mechanisms have been discussed.

WNT5A AND ITS RECEPTORS

The Wnt5a gene was first identified by Gavinet al.[10]and Nusse & Varmus[11]in mice,and mapped to chromosome 14.Subsequently,Clarket al.[12]identified the human Wnt5a gene on chromosome 3(3p14-p21).Wnt5a is a glycosylated protein that contains conserved cysteine (Cys77 and Cys104)[12]and serine residues (Ser209 and Ser244) that undergo palmitoylation,i.e.,covalent attachment to palmitate moieties,before binding to its frizzled receptors.[13]Ten frizzled (Fzs) seven-pass transmembrane proteins have been identified in mammals so far.Wnt5a binds to Fz3,Fz4,Fz5 and Fz8,and induces DSHl (disheveled 1) phosphorylation.[14]In addition,binding to Fz7 triggers activating protein 1 (AP-1) activation and Dishevelled (Dvl) polymerization,and that to Fz2 increases intracellular calcium concentration and activates Rac.[15]Thus,Wnt5a can regulate multiple cellular functionsviaFzs binding.

Tyrosine kinase-like orphan receptor 2 (Ror2),a single-pass transmembrane protein with a tyrosine kinase domain,is an essential receptor of Wnt5a.[16]The latter binds to Ror2 forming a ternary complex,which then interacts with Fzs and activates Wnt5a signaling.The transmembrane receptor-like tyrosine kinase (Ryk)is another essential Wnt5a receptor,[17]which binds to Wnt5a and other Wnts.[18]Some Wnts,like Wnt1a and Wnt3a,can bind to Fzs and low-density lipoprotein receptor-related protein co-receptor 5/6 (LRP5/6),initiating canonical Wnt signals.[19,20]In addition,Wnt-receptor binding inhibits the adenomatous polyposis coli(APC) and glycogen synthase kinase 3β (GSK3β) complex,which frees the cytoplasmic β-catenin that then binds to T-cell factor (TCF)/lymphoid enhancer factor(LEF),and activates the transcription of Wnt target genes.[21,22]However,Wnt5a also promotes β-catenin degradation,thus inhibites β-catenin-TCF/LEF binding and canonical Wnt genes transcription.[22](Figure 1)

Wnt5a regulates convergent extension movements by stimulating the Ca2+signaling pathway,[23]wherein the Wnt5a-Fzs complex activates recruitment of disheveled (DSH) proteins and phospholipase C(PLC),and mediates intracellular Ca2+release.[24-26]The increased Ca2+levels activate calmodulin dependent protein kinase Ⅱ (CAMK Ⅱ) or protein kinase C (PKC),leading to de-phosphorylation of nuclear factor of activated T cell (NFAT).[27,28]This pathway is crucial in regulating cell movement,proliferation and migration.[28]Wnt5a also regulates cellular gastrulation and maintenance via the planar cell polarity (PCP) pathway.[29]The Wnt5a-Ror2 complex can directly stimulate Jun N-terminal kinase (JNK),leading to uniform polarization,[30]as well as indirectly activate DSH and then the small GTPases Rho and Rac.[31,32](Figure 1)

WNT5A AND CANCER

Studies increasingly show different roles of Wnt5a in various cancers.Wnt5a-mediated non-classical pathway can antagonize the functions of the canonical Wnt/β-catenin pathway and inhibit cancer progression.[33,34]It also activates CAMK II,[35]siah2[36]or ROR2[37]to promote β-catenin degradation,and activates interleukin(IL)-10 upon Fzs binding to inhibit the TLR4-NF-kB signaling pathway.[38](Figure 2A) On the other hand,the Wnt5a/Ror2 signaling pathway can also promote cancer progression[39]via the pro-tumorigenic PI3K and PKC pathways.[40]Furthermore,Wnt5a triggers lamellipodia and filopodia formation,thus promoting cancer metastasis and invasion.[41](Figure 2B)

Gastric cancer and colorectal cancer

Wnt5a overexpression is observed in diffusive-type gastric cancer,[42,43]and is associated with tumor cell migration and invasion.It promotesin vitromigration of the gastric cancer SGC-7901 cell line via the GS3Kβ/PI3K/AKT pathway,[44]while antibody-mediated inhibition of Wnt5a suppresses the migration and invasion of the MKN-1,KKLS and TMK-1 cell lines.[45]Wnt5a overexpression promotes growth of KKLS and TMK-1 cells via laminin γ2,[46]induces Snail overexpression and upregulates CD113 in MKN-7 cells,[47]triggers epithelial-mesenchymal transition (EMT) in the SGC-7901 cells via Arf6-ERK signaling,[48]and promotes proliferation of MNK-45 cells by activating CXCL16–CXCR6 signaling.[49]Taken together,Wnt5a acts as an oncogene in gastric cancer and drives tumor progression.In contrast,Wnt5a overexpression indicates good prognosis in Dukes B colorectal cancer (CRC).[50]In addition,the highly metastatic CRC cell line SW620 shows low level of Wnt5a,which is increased upon inhibition of EMT and metastasis.[51]CRC patients with Wnt5a overexpress in the tumor tissues survive longer compared to those with Wnt5a loss,which may be linked to Wnt5a-mediated upregulation of 15-hydroxyprostaglandin dehydrogenase (15-PGDH).[52]Wnt5a also inhibits proliferation of the HCT116 and SW480 cell lines,[53]and is downregulated in the latter when EMT is stimulated by TGF-β.[54]However,one study showed that Wnt5a promoted CRC progression in the Apc1638N mouse model by stimulating cell proliferation and invasion.[55]These opposite effects might involve different WNT5A mRNA isoforms.For instance,Wnt5a-L mRNA inhibits proliferation of the HCT116 cells,whereas Wnt5a-S mRNA has a pro-growth effect.[56]

Non-small-cell lung cancer and breast cancer

Non-small-cell lung cancer (NSCLC) is a highly aggressive,metastatic disease with poor prognosis,[57]and Wnt5a overexpression has been observed in NSCLC patients.[58]Wnt5a down-regulation inhibits NSCLC cell invasion,migration and EMT,[59]while its up-regulation induced migration and clone formation of the H1975 cell line.[59]In NSCLC patients,Wnt5a expression is positively associated with that of stromal vascular endothelial growth factor,and might improve tumor neo-angiogenesis.[60]Wnt5a promotes malignant clone formation in cigarette smoking-related NSCLC,[61]and induces cisplatin-resistance in A549 cells by activating the PKC signaling pathway.[62]

The role of Wnt5a in breast cancer is more ambiguous.While some studies showed an inhibitory effect of Wnt5a,[63]and its overexpression in estrogen-receptor (ER) positive breast cancer patients indicated good prognosis.[63]Other studies have reported lower levels of Wnt5a in breast cancer tissues compared to normal tissues.[64]In addition,Wnt5a also inhibits migration and proliferation of the MDA-MB-231 and MDA-MB-468 cell lines by various mechanisms such as activating Cdc42,[65,66]reducing phosphofructokinase platelet-type(PFKP) expression,[33]splicing CD44 Mrna,[67]and downregulating CD44 expression levels.[64]However,there are reports of Wnt5a promoting invasion of the MDA-MB-231 cells by activating NF-κB and upregulating MMP-7 expression.[68]Furthermore,down-regulation of Wnt5a in the MDA-MB-175-VII cells inhibits their migration while Wnt5a overexpression increases malignancy of ER-positive breast cancer.[63]Finally,it induces the migration in MDA-MB-231 and MCF-7 cells by stimulating the Dvl2/Daam1/RhoA pathway.[69]In conclusion,Wnt5a has an overall pro-tumorigenic role in NSCLC and breast cancer,although the mechanisms need to be elucidated.

Pancreatic cancer and prostate cancer

Wnt5a overexpression in pancreatic cancer patients indicates poor prognosis and lymph node metastasis,[70]and promotes the proliferation and reduces apoptosis in PANC-1 and BXPC-3 cells.[71]In the murine pancreatic tumor model,Wnt5a overexpression induced EMT and cancer cell metastasis.[72]Furthermore,it promotes migration of the PANC1,Capan-2 and HT1080 cells by phosphorylating paxillin,[73]and induces gemcitabine resistance in PANC-1 and MIAPaCa2 cells via Cyclin D1 activation and AKT phosphorylation.[74]

Prostate cancer has a high rate of bone metastasis.In some patients,Wnt5a overexpression indicated poor prognosis.[75,76]It induces castration resistance in the LNCaP and 22Rv1 cell lines by stimulating BMP-6[75,77]and chemokine ligand 2 (CCL2) expression.[78]In the PC3,LNCaP,and DU145 cells,Wnt5a down-regulation can reduce proliferation and migration.[79]However,one study showed that Wnt5a overexpression in PC3,C42B,and MDA-PCa-2b cells inhibited their proliferation and induced apoptosis.[80]Furthermore,localized overexpression of Wnt5a in some prostate cancer patients indicates good prognosis.[81]In conclusion,Wnt5a has an oncogenic role in pancreatic cancer and a more complex function in prostate cancer,which likely depends on the downstream pathway.

Melanoma and leukemia

Wnt5a promotes melanoma progression,and high level of Wnt5a in melanoma patient is associated with poor prognosis.[82]In the melanoma cell line UACC 1273,Wnt5a overexpression inducesin vitroinvasion and proliferation by stimulating protein kinase C expression,[83]while blocking Wnt5a in the HTB63 and A375 cells reduced migration.[83]It also promotes the invasion and migration of the SKmel28,A2058,A375 and HTB63 cells by stimulating exosomes release,[84]and enhances chemo-resistance against BRAF inhibitors in A543 and MEL624 cells by activating AKT.[85]Thus,Wnt5a promotes melanoma progression by inducing cell invasion,migration,proliferation,and chemo-resistance.

In leukemia,the role of Wnt5a is ambiguous.While some studies show that Wnt5a overexpression is a protective factor in acute lymphoblastic leukemia,[86]and Wnt5a silence is observed in acute leukemia;[87]other studies have illustrated that Wnt5a overexpression promotes leukemia cells proliferation and migration via inducing Ror1 and Ror2.[88,89]These opposite effects might involve Wnt5a epigenetic changes.For instance,hypermethylation of WNT5A promotor region is observed in acute lymphoblastic leukemia and it leads to downregulation of WNT5A mRNA level.[90]In acute leukemia,histone H4K20me1 is enriched in WNT5A promotor and coding region,regulating Wnt5a expression via inducing transcription elongation and initiation.[91]

CONCLUSION

Wnt5a has an ambiguous role in tumorigenesis,as indicated by the conflicting reports in different cancer types.Wnt5a can influence on cell proliferation,invasion,migration,metastasis,and chemo-resistance.While it promotes tumor progression in gastric cancer,NSCLC,pancreatic cancer and melanoma,Wnt5a has opposite effects in colorectal cancer,breast cancer and prostate cancer,which likely involves distinct signaling pathways.Furthermore,in colorectal cancer and leukemia,distinct WNT5A isoforms and epigenetic modification lead to different roles of Wnt5a.WNT5A DNA hypermethylation and histone methylation might influence on WNT5A mRNA isoforms and Wnt5a protein expression,leading to different Wnt5a expression in cancers.Furthermore,Wnt5a regulates various signal pathways and these pathways have distinct effects on cancer progression (Figure 3).In conclusion,WNT5A methyl modification and Wnt5a protein effects on various signal pathways might contribute to its controversial roles in cancers.

Conflict of interests

All authors declared no conflicting interests.