The role of WNT5A in oral squamous cell carcinoma

Abstract: Cancer is one of the leading causes of death worldwide and furtherresearch into the cancer biology is required to improve treatment andsurvival. Cancer can start in all sites of the body and is characterizedby uncontrolled cell growth and capability of invading surroundingtissue and spreading to other sites of the body. The most commontype of cancer occurring in the oral cavity is oral squamous cellcarcinoma (OSCC). Tobacco, including the betel quid and othertypes of smokeless tobacco, is the main risk factor for developmentof OSCC. Even though therapeutic treatment of OSCC has beenimproved, the prognosis is still poor, and the 5-year survival remainsaround 50%. The ongoing research have shown that proteins involvedin signaling pathways play an important role in the progression ofcancer, however, no reliable biomarkers have yet been identified aspredictors of OSCC progression.Over the past years, a protein named WNT5A, has been shownto be involved in different types of cancer, either by promoting orsuppressing cancer progression. However, its role in OSCC is stillambiguous. This thesis aimed to provide an insight into the role ofWNT5A signaling in OSCC.We started by investigating the functional role of WNT5A in OSCCcells. Due to the lack of the endogenous WNT5A expression, wetreated the cells with recombinant WNT5A (rWNT5A) and observedactivation of the non-canonical WNT/Ca2+/PKC signaling in OSCCcells. This rWNT5A-induced signaling enhanced migration andinvasion of OSCC cells, which was ascertained by different WNT5Ainhibitors. These inhibitors eliminated the rWNT5A-induced WNT/Ca2+/PKC signaling and migration of OSCC cells indicating apromoting role of WNT5A in OSCC cells.Before investigating the expression of WNT5A in human OSCCtissues, we evaluated four commercially available WNT5A antibodiesfor use in immunohistochemistry (IHC) and western blot analysis(WB). Cytoplasmic WNT5A immunostaining pattern was observedwith all four antibodies but only the polyclonal AF645 antibody wasable to detect WNT5A protein in WNT5A-positive cell lysates. Preabsorptiontests revealed that the polyclonal AF645 antibody is thebest antibody for detection of WNT5A by WB while the monoclonal3A4 antibody is the most suitable for use in IHC. Using the monoclonal 3A4 antibody, we investigated the expressionof WNT5A in human OSCC tissues. No expression of WNT5A wasobserved in normal oral epithelium or in mild grade of dysplasia.However, cytoplasmic WNT5A immunostaining was found in 38%of dysplastic tissues and in 81% of the OSCCs. We also noticedthat WNT5A was more expressed in advanced OSCCs than in earlyinvasive OSCCs. Furthermore, we did not observe any correlationbetween expression of WNT5A and two adhesion-proteins, β-cateninand E-cadherin, either in OSCC tissues or in OSCC cells. Thesefindings suggest that WNT5A does not affect the canonical WNT/β-catenin pathway or downregulation of E-cadherin in OSCC.At last, we investigated if the rWNT5A-induced WNT/Ca2+/PKCsignaling affected secretion and activation of matrix metalloproteinases(MMPs) in OSCC cells. We found that rWNT5A-induced activationof MMP2 is not dependent on activation of either β-catenin, ERK1/2,or p38-MAPK, but could instead be induced by WNT5A/Ca2+/PKCpathway. In conclusion, these findings suggest that WNT5A acts as a cancerpromoter in OSCC by facilitating cell migration and invasion via theWNT5A/Ca2+/PKC signaling and activation of MMP2.