SUBMITTED 8 AUG 22 1 REVISION REQ. 13 SEP 22; REVISION RECD. 4 OCT 22 2 ACCEPTED 1 NOV 22 3 ONLINE-FIRST: DECEMBER 2022 4 DOI: https://doi.org/10.18295/squmj.12.2022.066 5 6 Expression of Dkk 1 in Endometrial Endometrioid Carcinoma & Its Correlation 7 with Wnt / β-catenin Signaling Pathway 8 Abhijit Das,1 *Sandeep R. Mathur,2 Sunesh Kumar,3 Neerja Bhatla3 9 10 1Department of Pathology, Janakpuri Super Speciality Hospital, New Delhi, India; Departments 11 of 2Pathology and 3Obstetrics and Gynaecology, All India Institute of Medical Sciences, New 12 Delhi, India. 13 *Corresponding Author’s e-mail: mathuraiims@gmail.com 14 15 Abstract 16 Objective: Endometrial cancer is the most common form of cancer affecting female reproductive 17 organs. Most common histologic type endometrioid carcinoma constitutes 75 to 80% of all cases. 18 Studies on Dkk1 expression profiles and its inhibitory role in Wnt signaling pathway in genesis 19 and development of endometrial carcinoma are very few. This study aims to investigate Dkk1 20 expression in endometrial carcinoma and its correlation with Wnt/β-catenin pathway. Methods: 21 A total of 160 formalin fixed paraffin embedded samples including 50 cases each of endometrial 22 atypical hyperplasia and endometrioid endometrial carcinoma along with 30 cases each of 23 proliferative and secretory endometrium were included in this study. We investigated expression 24 pattern of Dkk1, E-cadherin, β-catenin and c-myc in endometrial atypical hyperplasia and 25 carcinoma as well as compared with that of proliferative and secretory endometrium. 26 Immunohistochemistry and analysis were performed from July, 2018 to June, 2020. Results: We 27 showed decreasing pattern of immunopositivity for Dkk1, E-cadherin and β-catenin from 28 proliferative/secretory endometrium to endometrial atypical hyperplasia and endometrioid 29 carcinoma. Increasing c-myc immunopositivity was noted from proliferative/secretory 30 endometrium to endometrial atypical hyperplasia and endometrioid carcinoma. Moreover, 31 decreasing Dkk1 immunopositivity was well correlated with both E-cadherin, β-catenin and c-32 myc immunopositivity. Conclusion: Decreasing Dkk1 positivity from benign endometrium to 33 endometrioid carcinoma suggests a negative regulatory function of Dkk1 in endometrioid 34 carcinoma. Dkk1 is downregulated in Wnt signaling pathway in endometrioid endometrial 35 carcinoma. Thus, Dkk1 can show promise as a biomarker for screening endometrioid carcinoma. 36 Future researches can study the reactivation of the Dkk1 gene that could be a valuable strategy 37 for antagonizing Wnt signaling pathway. 38 Keywords: Endometrioid carcinoma, Dkk1, Wnt/β-catenin pathway, β-catenin, E-cadherin 39 40 Advances in Knowledge 41  Dkk1 shows decreasing trend of immunoexpression from benign phase endometrium to 42 endometrioid endometrial carcinoma. 43  Expression of Dkk1 is well correlated with the markers (β-catenin, E-cadherin, c-myc) of 44 Wnt signaling pathway. 45  Dkk1 has an antagonistic role in Wnt signaling pathway. 46 47 Application to Patient Care 48  Dkk1 can be a promising biomarker in screening progression of endometrioid 49 endometrial carcinoma. 50  Reactivation of Dkk1 gene could be a valuable strategy to antagonize Wnt signaling 51 pathway in endometrioid endometrial carcinoma. 52 53 Introduction 54 Endometrial cancer is the most prevalent invasive gynecologic malignancy among American 55 women accounting for 7% of estimated new cancer cases in 2021.1 Incidence and death rates of 56 endometrial cancer have been increasing by an average of 1.1% and 0.3% per year respectively.2 57 The most common histological type, endometrioid adenocarcinoma constitutes 75-80% of 58 endometrial cancers. The disease mostly affects postmenopausal women with an average age of 59 60 years at diagnosis, while in women younger than 40 years it constitutes only five percent.3 In 60 India it ranks third among female genital tract malignancies, after carcinoma cervix and 61 carcinoma ovary.4 Most of the cases are diagnosed in early stages because of abnormal uterine 62 bleeding. The best diagnostic strategy in postmenopausal patient presenting with abnormal 63 uterine bleeding, still remains controversial. Nowadays, endometrial biopsy and hysteroscopy 64 have almost replaced dilatation and curettage (D&C) for the diagnosis and management of 65 endometrioid carcinoma.5 Recent studies showed that the first step in the diagnostic pathway 66 should be the measurement of endometrial thickness, followed by endometrial sampling.6 67 Clinical assessment, radiological evaluation and histopathological examination have led the way 68 to study of molecular pathways like Wnt signaling pathway. Wnt signal transduction pathway is 69 activated by binding of a Wnt protein to cell surface receptor. E-cadherin (a cell adhesion 70 molecule forming adherens junctions between cells), β-catenin (a subunit of cadherin protein 71 complex) and c-myc (a transcription factor protein regulating cell proliferation) are integral 72 components of Wnt signaling pathway. Abnormalities of Wnt signaling transduction pathway 73 [Figure 1] is responsible for genesis and development of some human malignant tumors.7 74 Attempts have been made to investigate various regulators in the Wnt signaling pathway as 75 targets for diagnosis and treatment of malignant tumors. Several candidate markers, such as E-76 cadherin, β-catenin, c-myc and others have been proposed for use on cytologic or histologic 77 samples in endometrial carcinoma.8 As a negative regulator in Wnt signaling pathway, Dkk1 can 78 inhibit Wnt activation in tumor progression.9,10 Earlier studies in colorectum and placenta 79 showed that Dkk1 was prominently expressed in normal cells but absent in cancer cells.11 At 80 present, studies on Dkk1 expression profiles in endometrial carcinoma are very few.12 Dkk1 81 expression pattern in endometrial carcinoma and its correlation with other components of Wnt 82 pathway, especially β-catenin, E-cadherin and c-myc has not been studied so far in India. This 83 study will investigate the expression pattern of Dkk1, E-cadherin, β-catenin and c-myc in 84 endometrial carcinoma. Moreover, the expression pattern of these markers in endometrial 85 atypical hyperplasia and carcinoma will be compared with that of proliferative and secretory 86 endometrium. 87 88 Methods 89 Selection of Cases 90 This retrospective study was conducted at the Department of Pathology where formalin fixed 91 paraffin embedded (FFPE) samples of endometrial lesions, age ranging from 21 to 77 years, 92 collected between January 2005 and March 2018, were selected including 50 cases each of 93 endometrial atypical hyperplasia and endometrioid endometrial carcinoma along with 30 cases 94 each of proliferative and secretory endometrium. Endometrial samples in younger patients were 95 taken primarily to exclude the causes of infertility and abnormal uterine bleeding. Standard 96 morphological criteria were used for diagnosis and selection of cases and control groups. The 97 study was approved by the Institutional Ethics Committee. One section of each sample was 98 stained with hematoxylin and eosin (H&E) and four step sections on coated slides were used for 99 Dkk1, E-cadherin, β-catenin and c-myc immunohistochemistry (IHC). Immunohistochemistry 100 and analysis were performed over the next 2 years from July, 2018 to June, 2020. 101 102 Immunohistochemistry 103 Immunohistochemistry was performed using available monoclonal antibodies for Dkk1, E-104 cadherin, β-catenin and c-myc (Dkk1, Abcam, 1:100; β-catenin, Thermo Scientific, 1:400; c-105 myc, Thermo Scientific, 1:100; and E-cadherin, Thermo Scientific, 1:200). 106 107 Steps. Serial 4-micron thick sections were cut from the selected representative paraffin 108 embedded tissue blocks and 3-aminopropyl triethoxysilane (APTES) coated slides were used for 109 IHC. Slides were deparaffinized, followed by rehydration in decreasing concentration of alcohol. 110 For Dkk1, E-cadherin and c-myc immunostains, antigen retrieval was done by heating the 111 sections in citrate buffer inside a 600 watt microwave oven at full power for 30 minutes. For β-112 catenin Tris-EDTA buffer at pH 8 was used for heat mediated antigen retrieval. To diminish the 113 nonspecific immunostaining (i.e. endogenous peroxidase activity), each slide was treated with 114 methanol containing 4% hydrogen peroxide for 30 minutes. For all immunostains, sections were 115 then overlaid with adequate amount of appropriately diluted primary antibody followed by 116 overnight incubation at 40C in a humid chamber. After 3 changes of washing (5 minutes each) in 117 Tris- HCl buffer peroxidase conjugated streptavidin was applied to cover the sections and 118 incubated at room temperature for 30 minutes. Each section was then covered with substrate 119 chromogen solution freshly prepared by dissolving 50 µl of Di-amino Benzidine (DAB) 120 chromogen to 1 ml of DAB substrate buffer. The sections were counterstained with hematoxylin 121 for 10 seconds, followed by mounting with DPX. During staining of each bach, appropriate 122 positive and negative controls (by omitting primary antibody) were used. 123 124 Analysis. IHC stains (Dkk1, cytoplasmic; β-catenin, membranous; c-myc, cytoplasmic and 125 nuclear; E-caderin, membranous) were reviewed and analysed in conjunction with hematoxylin 126 and eosin (H&E) stained slides. Immunoreactive score (IRS) was obtained by multiplying 127 intensity score (0, no staining; 1, weak; 2, moderate and 3 strong staining) and percentage score 128 (0, nil; 1, <10%; 2, 10-50%; 3, 51-80% and 4, >80%). Thus, the total IRS score ranged from 0 to 129 12.13 Two independent observers had analyzed the expression pattern of all four markers and 130 then an average was calculated for final analysis. Appropriate statistical tests including 131 independent sample t test, Chi-square test and Pearson correlation test were applied to analyze 132 the significance of results between cases and control groups using the Statistical Package for the 133 Social Sciences (SPSS), version 21.0 (IBM Inc., Chicago, Illinois, USA) software program. The 134 P<0.05 was considered statistically significant. 135 136 Results 137 The retrospective study evaluated a total number of 160 samples including proliferative 138 endometrium, secretory endometrium, atypical hyperplasia and endometrioid carcinoma. 139 Immunoprofiles using Dkk1, E-cadherin, c-myc and β-catenin were compiled, compared and 140 analyzed for different expression pattern in various groups of endometrium. 141 142 Age Distribution. Age pattern of proliferative group versus secretory group was statistically 143 insignificant (P value 1.000), while the age patterns between proliferative endometrium versus 144 endometrial atypical hyperplasia; proliferative endometrium versus endometrial carcinoma; 145 secretory endometrium versus endometrial atypical hyperplasia; secretory endometrium versus 146 endometrial carcinoma; as well as endometrial atypical hyperplasia versus endometrial 147 carcinoma were statistically significant (P value <0.001). 148 149 Intergroup Dkk1 Immunopositivity. Dkk1 showed mostly cytoplasmic expression in glandular 150 epithelium during proliferative phase, endometrial atypical hyperplasia and endometrioid 151 carcinoma. However, 2 cases of proliferative endometrium had nonspecific nuclear positivity 152 both in glandular epithelium and the stroma. Secretory endometrium showed cytoplasmic 153 immunopositivity both in glandular as well as stromal cells. Squamous morules associated with 154 endometrioid carcinoma also had similar cytoplasmic immunopositivity. We have studied 155 cytoplasmic expression among the groups. Dkk1 immunopositivity of proliferative endometrium 156 versus secretory endometrium was statistically insignificant (P value 0.183). There was 157 increased Dkk1 immunopositivity in proliferative endometrium as compared to endometrial 158 atypical hyperplasia and endometrioid carcinoma [Figure 2], which was statistically significant 159 (P value <0.001). Dkk1 immunopositivity of endometrial atypical hyperplasia versus 160 endometrioid carcinoma was statistically insignificant (P value 1.000). Secretory endometrium 161 showed increased Dkk1 immunopositivity as compared to endometrial atypical hyperplasia and 162 endometrioid carcinoma and the difference was statistically significant (P value <0.001). Dkk1 163 showed decreasing trend of expression from endometrial atypical hyperplasia to grade I 164 endometrioid carcinoma to grade II endometrioid carcinoma. When individual grades are 165 compared separately, the difference between endometrial atypical hyperplasia and grade I 166 endometrioid carcinoma was statistically insignificant (P value 1.000), but it was statistically 167 significant in between endometrial atypical hyperplasia and grade II endometroid carcinoma (P 168 value 0.048). 169 170 Intergroup E-cadherin Immunopositivity. E-cadherin showed membranous immunopositivity. E-171 cadherin immunopositivity of proliferative endometrium versus secretory endometrium was 172 statistically insignificant (P value 1.000). Immunopositivity of both proliferative endometrium 173 and secretory endometrium were higher than that of endometrial atypical hyperplasia and 174 endometrioid carcinoma [Figure 3]; and the difference in immunopositivity among them were 175 statistically significant (P value <0.001). There was also statistically significant difference 176 between endometrial atypical hyperplasia and endometrioid carcinoma (P value <0.001). 177 178 Intergroup β-catenin Immunopositivity. Membranous β-catenin expression was studied among 179 the groups. Nuclear β-catenin was observed in 14% (7/50) of endometrioid carcinoma excluding 180 the areas of squamous morule formation that also showed nuclear positivity. β-catenin 181 immunopositivity of proliferative endometrium versus secretory endometrium was statistically 182 insignificant (P value 1.000). In this study, both proliferative endometrium and secretory 183 endometrium showed increased immunopositivity of β-catenin as compared to endometrial 184 atypical hyperplasia and endometrioid carcinoma [Figure 4]; and the difference in β-catenin 185 immunopositivity among them were statistically significant (P value <0.001). Immunopositivity 186 in endometrial atypical hyperplasia was statistically significant (P value <0.001) when compared 187 to that of endometrial carcinoma. 188 189 Intergroup c-myc Immunopositivity. We evaluated cytoplasmic c-myc immunopositivity among 190 the groups. Additionally, nuclear expression was noted in 14 cases and 4 cases of proliferative 191 and secterory endometrium respectively. When c-myc immunopositivity of proliferative 192 endometrium versus secretory endometrium was compared, the difference was statistically 193 insignificant (P value 1.000). There was increased immunopositivity in endometrioid carcinoma 194 as compared to proliferative endometrium and endometrial atypical hyperplasia [Figure 5]; the 195 difference in c-myc immunopositivity among them were statistically significant (P value 0.043 196 and <0.001 respectively). By contrast, c-myc immunopositivity of secretory endometrium versus 197 endometrial atypical hyperplasia was statistically insignificant (P value 0.384), while c-myc 198 immunopositivity of secretory endometrium versus endometrioid carcinoma was statistically 199 significant (P value <0.001). 200 201 Intragroup Correlation among Immunohistochemistry Markers. In endometrial atypical 202 hyperplasia group, we found statistically significant correlation between Dkk1 and β-catenin 203 immunopositivity, as well as between E-cadherin and c-myc immunopositivity. Rest three 204 groups didn’t show any significant correlation among the four IHC markers. Comparison of 205 immunohistchemistry between two age groups in endometrial atypical hyperplasia and 206 endometrioid carcinoma as well as between grade I and grade II endometrioid carcinomas didn’t 207 reveal any significant difference [Table 1]. 208 209 Discussion 210 Endometrial cancer has surpassed cervical cancer as the most common gynecologic malignancy. 211 Cervical cancer was much more prevalent in past few decades compared to endometrial cancer, 212 but earlier detection and eradication of cervical precursor lesions has reversed the ratio.14 213 Endometrial carcinoma frequently occurs in peri-and post-menopausal women with 214 endometrioid carcinoma being the most common histological subtype.3,15 PTEN genetic 215 mutation is most frequent (39-83%) in endometrioid cancer, however β-catenin mutation 216 accounts for 31-47% of the cases.16 β-catenin is an integral component of Wnt signaling 217 pathway [Figure 1], that is dysregulated in many human cancers. On contrary, a negative 218 regulator of β-catenin pathway, Dkk1 prevents tumor progression by inhibiting this signaling 219 pathway.9 Some studies described role of Dkk1 in non-endometrial tissues both in normal and 220 corresponding malignant cells, however studies on endometrial cancer are very less in English 221 literature.11,12 Hence, we have tried to evaluate expression pattern of Dkk1 in various groups of 222 benign, atypical and malignant endometrium as well as correlated with other markers like E-223 cadherin, β-catenin, c-myc of Wnt pathway to show their relation among the groups. 224 225 Dkk1. Dkk1 is a glycoprotein and one of the members of Dkk family (Dkks), secreted by various 226 cells throughout the human body.17 The human Dkk1 gene maps to chromosome 10q11.2, 227 which encodes a protein that acts as an antagonist in Wnt signaling pathway [Figure 1C] by 228 binding to and inhibiting LRP 5/6.18 Yi N et al showed Dkk1 positivity both in benign 229 endometrium and endometrial carcinoma, where Dkk1 was mostly distributed in the cytoplasm 230 of glandular epithelium. They have documented ‘high expression’ of Dkk1 predominantly in 231 benign endometrium, in contrast to “low expression” in endometrial cancer suggesting that this 232 reduction expression may be due to its negative regulatory function in Wnt signaling pathway.12 233 We also found decreasing Dkk1 immunopositivity from proliferative/secretory endometrium to 234 endometrial atypical hyperplasia and endometrioid carcinoma. In our study Dkk1 positivity was 235 predominantly in the cytoplasm of glandular epithelium, however stromal cells also showed 236 weak cytoplasmic immunopositivity [Figure 2]. We also found significant difference in Dkk1 237 immunopositivity between endometrial atypical hyperplasia and proliferative/secretory 238 endometrium; as well as between endometrioid carcinoma and proliferative/secretory 239 endometrium. Though there was increased Dkk1 immunopositivity in endometrial atypical 240 hyperplasia as compared to endometrioid carcinoma, it did not achieve statistical significance. 241 Interestingly some studies demonstrated reduced expression of β-catenin following treatment 242 with exogenous Dkk1 probably indicating that increased Dkk1 binding to LRP5/6 inhibits Wnt 243 signaling leading to degradation of β-catenin.19 Decreasing Dkk1 positivity in our study from 244 benign endometrium to endometrioid carcinoma may suggest that negative regulatory function of 245 Dkk1 is reduced from benign to malignant endometrium. Thus at least in part, by inducing 246 abnormalities of Wnt signaling pathway, Dkk1 plays a role in the genesis and development of 247 endometrial carcinoma. Similar patterns of Dkk1 alterations have also been reported in some 248 other tumors including colorectal cancer, placental choriocarcinoma and non-small cell lung 249 cancers where Dkk genes were frequently silenced.11,20 In our study decreasing positivity of 250 Dkk1 from proliferative/secretory endometrium to endometrial atypical hyperplasia and 251 endometrioid carcinoma, suggests that Dkk1 is involved in the early phase of endometrioid 252 carcinoma by suppressing Wnt pathway. 253 254 E-cadherin. Cell surface glycoprotein E-cadherin with a molecular weight of 120 kDa is a major 255 cadherin molecule expressed by epithelial cells. It binds to catenin [Figure 1A] to form a 256 cadherin-catenin complex that plays an important role in intercellular adhesion.21 Shih et al 257 demonstrated that the cytoplasmic expression of E-cadherin in endometrial glandular cells 258 occurred mainly in the proliferative phase and decreased in the secretory phase.7 In contrast to 259 this study we found strong membranous immunopositivity both in proliferative and secretory 260 endometrium. Although, similar to their study, we found decreased E-cadherin expression in 261 endometrioid carcinoma as compared to proliferative/secretory endometrium. The mechanism of 262 reduced of E-cadherin positivity has not been fully understood, however, Saito et al showed that 263 loss of E-cadherin positivity was caused by promoter methylation of the E-cadherin gene.22 In 264 our study, we found significant difference in E-cadherin immunopositivity between endometrial 265 atypical hyperplasia and proliferative/secretory endometrium; as well as between endometrioid 266 carcinoma and proliferative/secretory endometrium. We also showed that E-cadherin 267 immunopositivity was significantly different between endometrial atypical hyperplasia and 268 endometrioid carcinoma. So far, none of the previous studies has mentioned difference in E-269 cadherin positivity between endometrial atypical hyperplasia and carcinoma. 270 271 β-catenin. β-catenin encoded by CTNNB1 gene is a subunit of the cadherin protein complex. It 272 takes part in the formation of adherens junctions [Figure 1], that plays a pivotal role in 273 maintaining epithelial cell layers by regulating cellular adhesion and growth signals.23 Several 274 studies showed that it has been implicated in the pathogenesis and progression of many human 275 malignancies involving Wnt pathway. As a signal transducer in Wnt pathway it induces targeted 276 gene expression and cytoplasmic β-catenin accumulation.24 Previous studies demonstrated 277 greater positivity of cytoplasmic β-catenin in the glandular cells of proliferative endometrium as 278 compared to secretory phase. These studies also showed nuclear positivity of β-catenin in the 279 glandular cells of the proliferative and early secretory phase endometrium.7,24 However, we did 280 not find any difference in β-catenin immunopositivity between proliferative and secretory 281 endometrium as well as no nuclear β-catenin immunopositivity in proliferative/secretory 282 endometrium or in endometrial atypical hyperplasia. Shih et al revealed that the nuclear β-283 catenin-positive cells lacked E-cadherin positivity which indicated an inverse correlation 284 between E-cadherin and nuclear β-catenin positivity.7,25 This result was concordant with our 285 study where 14% of endometrioid carcinoma showed nuclear β-catenin immunopositivity, and 286 most of them showed near total loss of membranous E-cadherin immunopositivity. Exact 287 mechanisms behind this reduced positivity of E-cadherin at nuclear β-catenin positive sites are 288 still not elucidated, however it may be due to nuclear translocation of β-catenin that impairs the 289 β-catenin/E-cadherin adherent junction complex that finally leads to E-cadherin release from the 290 cell membrane. 291 292 The mechanisms of nuclear accumulation of β-catenin are reported to be responsible for the 293 mutation of β-catenin and related genes. Studies on Wnt pathway in colorectal cancers 294 demonstrated β-catenin stabilization and its significant accumulation in the cell which were 295 primarily attributed to the mutation of the adenomatosis polyposis coli (APC) or β-catenin gene 296 in the signaling pathway resulting in cell cycle progression in colorectal cancer.26 Our study 297 showed decreasing membranous immunopositivity of β-catenin from proliferative/secretory 298 endometrium to endometrial atypical hyperplasia and endometrioid carcinoma. We also showed 299 that there was significant difference in β-catenin immunopositivity between endometrial atypical 300 hyperplasia and proliferative/secretory endometrium; between endometrioid carcinoma and 301 proliferative/secretory endometrium; as well as between endometrial atypical hyperplasia and 302 endometrioid carcinoma. In this study, nuclear β-catenin positive cases of endometrioid 303 carcinoma showed increased cytoplasmic c-myc immunopositivity. Hence, both c-myc and β-304 catenin were found to be upregulated in these cases of endometrioid carcinomas. 305 306 c-myc. c-myc is a nuclear DNA binding protein that is implicated in cell cycle regulation. c-myc 307 amplifications in many human cancers were found to be associated with tumor aggressiveness 308 and poor prognosis.27 A cyclic variation in the c-myc positivity was reported by Odom et al with 309 higher expression in the proliferative than in the secretory phase.28 In contrast to this finding, we 310 observed increased c-myc immunopositivity in secretory endometrium as compared to 311 proliferative endometrium. Bircan et al in their study showed that the anti c-myc monoclonal 312 antibody was detected both in the nucleus and the cytoplasm, which was concordant with our 313 study. Actively dividing cells of proliferative phase endometrium displayed a nuclear 314 distribution, while in differentiated cells of the secretory phase the immunostaining was 315 primarily cytoplasmic.29 They showed cytoplasmic and perinuclear c-myc positivity in 15.3% of 316 endometrial cancers. Another study by Geisler et al demonstrated both cytoplasmic and nuclear 317 c-myc immunopositivity in 75.2% and 66.9% of cases of endometrial cancers respectively.30 By 318 contrast, we found only cytoplasmic c-myc immunopositivity in all cases of endometrioid 319 carcinomas along with few cases of proliferative and secretory endometrium showing nuclear c-320 myc immunopositivity. We also found increasing cytoplasmic immunopositivity of c-myc from 321 proliferative/secretory endometrium to endometrial atypical hyperplasia and endometrioid 322 carcinoma. There was also significant difference in immunopositivity between endometrial 323 atypical hyperplasia and proliferative endometrium; between endometrioid carcinoma and 324 proliferative/secretory endometrium; as well as between endometrial atypical hyperplasia and 325 carcinoma. However, we did not find any significant difference in c-myc immunopositivity 326 between endometrial atypical hyperplasia and secretory endometrium. 327 328 Conclusion 329 Decreasing Dkk1 immunopositivity from proliferative/secretory endometrium to endometrial 330 atypical hyperplasia to endometrioid carcinoma indicates that Dkk1 is downregulated in 331 endometrioid endometrial carcinoma. Immunoprofiles of Dkk1 and the other markers associated 332 with Wnt signaling pathway explain the antagonistic role of Dkk1 in the Wnt signaling pathway 333 in endometrial cancer. Thus, Dkk1 shows promise as a biomarker for screening progression of 334 endometrioid carcinoma. On the other hand, reactivation of the Dkk1 gene could be a valuable 335 strategy for antagonizing Wnt signaling pathway. 336 337 Conflicts of Interest 338 The authors declare no conflict of interests. 339 340 Funding 341 No funding was received for this study. 342 343 Author Contributions 344 AD and SM conceptualised and designed the study. SM drafted the manuscript. SK and NB 345 performed critical review and contributed with suggestions. SM and AD were involved in data 346 collection, data entry, literature search, and data analysis. All the authors approved the final 347 version of the manuscript. 348 349 References 350 1. American Cancer Society. Cancer Facts and Figures 2021. Atlanta, Ga: American Cancer 351 Society, 2021. Available online Last accessed November 23, 2021. 352 https://doi.org/10.1057/978-1-349-96042-2_16551. 353 2. Lewis DR, Chen HS, Cockburn MG, Wu XC, Stroup AM, Midthune DN, Zou Z, Krapcho 354 MF, Miller DG, Feuer EJ. Early estimates of SEER cancer incidence, 2014. Cancer 2017; 355 123:2524-2534. https://doi.org/10.1002/cncr.30630. 356 3. Kushner DM, Lurain JR, Fu TS, Fishman DA. Endometrial adenocarcinoma metastatic to the 357 scalp: case report and literature review. Gynecol Oncol 1997; 65:530-3. 358 https://doi.org/10.1006/gyno.1997.4718. 359 4. Nath, A., & Mathur, P. (2021). Strengthening Cancer Surveillance in India: Role of the 360 National Cancer Registry Programme. Indian Journal of Surgical Oncology. 361 https://doi.org/10.1007/s13193-021-01473-8. 362 5. Colombo N, Preti E, Landoni F, Carinelli S, Colombo A, Marini C, Sessa C; ESMO 363 Guidelines Working Group. Endometrial cancer: ESMO Clinical Practice Guidelines for 364 diagnosis, treatment and follow-up. Ann Oncol 2011; 22 Suppl 6:vi35-9. 365 https://doi.org/10.1093/annonc/mdr374. 366 6. Vergote I, Amant F, Timmerman D. Should we screen for endometrial cancer? Lancet Oncol 367 2011; 12:4-5. https://doi.org/10.1016/s1470-2045(10)70280-1. 368 http://www.cancer.org/acs/groups/content/@epidemiologysurveilance/documents/document/acspc-031941.pdf 7. Bienz M, Clevers H. Linking colorectal cancer to Wnt signaling. Cell 2000; 103:311-20. 369 https://doi.org/10.1016/s0092-8674(00)00122-7. 370 8. Shih HC, Shiozawa T, Miyamoto T, Kashima H, Feng YZ, Kurai M, Konishi I. 371 Immunohistochemical expression of E-cadherin and beta-catenin in the normal and 372 malignant human endometrium: an inverse correlation between E-cadherin and nuclear beta-373 catenin expression. Anticancer Res 2004; 24:3843-50. PMID: 15736420. 374 9. Logan CY, Nusse R. The Wnt signaling pathway in development and disease. Annu Rev Cell 375 Dev Biol 2004; 20:781-810. https://doi.org/10.1146/annurev.cellbio.20.010403.113126. 376 10. Mao J, Wang J, Liu B, Pan W, Farr GH 3rd, Flynn C, Yuan H, Takada S, Kimelman D, Li L, 377 Wu D. Low-density lipoprotein receptor-related protein-5 binds to Axin and regulates the 378 canonical Wnt signaling pathway. Mol Cell 2001; 7:801-9. https://doi.org/10.1016/s1097-379 2765(01)00224-6. 380 11. Peng S, Miao C, Li J, Fan X, Cao Y, Duan E. Dickkopf-1 induced apoptosis in human 381 placental choriocarcinoma is independent of canonical Wnt signaling. Biochem Biophys Res 382 Commun 2006; 350:641-7. https://doi.org/10.1016/j.bbrc.2006.09.087. 383 12. Yi N, Liao QP, Li T, Xiong Y. Novel expression profiles and invasiveness-related biology 384 function of DKK1 in endometrial carcinoma. Oncol Rep 2009; 21:1421-7. 385 https://doi.org/10.3892/or_00000370. 386 13. Pyo SW, Hashimoto M, Kim YS, Kim CH, Lee SH, Johnson KR, Wheelock MJ, Park JU. 387 Expression of E-cadherin, P-cadherin and N-cadherin in oral squamous cell carcinoma: 388 correlation with the clinicopathologic features and patient outcome. J Craniomaxillofac Surg 389 2007; 35:1-9. https://doi.org/10.1016/j.jcms.2006.11.004. 390 14. Kumar V, Abbas AK, Aster JC: Robbins and Cotran Pathologic Basis of Disease. 9th Ed. 391 Elsevier Saunders, Philadelphia, 2015. P.1014. 392 15. Bokhman JV. Two pathogenetic types of endometrial carcinoma. Gynecol Oncol 1983; 393 15:10-7. https://doi.org/10.1016/0090-8258(83)90111-7. 394 https://doi.org/10.1016/s0092-8674(00)00122-7 16. Bansal N, Yendluri V, Wenham RM. The molecular biology of endometrial cancers and the 395 implications for pathogenesis, classification, and targeted therapies. Cancer Control 2009; 396 16:8-13. https://doi.org/10.1177/107327480901600102. 397 17. Bafico A, Liu G, Yaniv A, Gazit A, Aaronson SA. Novel mechanism of Wnt signalling 398 inhibition mediated by Dickkopf-1 interaction with LRP6/Arrow. Nat Cell Biol 2001; 3:683-399 6. https://doi.org/10.1038/35083081. 400 18. Mao B, Wu W, Li Y, Hoppe D, Stannek P, Glinka A, Niehrs C. LDL-receptor-related protein 401 6 is a receptor for Dickkopf proteins. Nature 2001; 411:321-5. 402 https://doi.org/10.1038/35077108. 403 19. Maniatis T. A ubiquitin ligase complex essential for the NF-kappaB, Wnt/Wingless, and 404 Hedgehog signaling pathways. Genes Dev 1999; 13:505-10. 405 https://doi.org/10.1101/gad.13.5.505. 406 20. Licchesi JD, Westra WH, Hooker CM, Machida EO, Baylin SB, Herman JG. Epigenetic 407 alteration of Wnt pathway antagonists in progressive glandular neoplasia of the lung. 408 Carcinogenesis 2008; 29:895-904. https://doi.org/10.1093/carcin/bgn017. 409 21. Fadare O, Reddy H, Wang J, Hileeto D, Schwartz PE, Zheng W. E-Cadherin and beta-410 Catenin expression in early stage cervical carcinoma: a tissue microarray study of 147 cases. 411 World J Surg Oncol 2005; 3:38. https://doi.org/10.1186/1477-7819-3-38. 412 22. Saito T, Nishimura M, Yamasaki H, Kudo R. Hypermethylation in promoter region of E-413 cadherin gene is associated with tumor dedifferention and myometrial invasion in 414 endometrial carcinoma. Cancer 2003;97:1002-9. https://doi.org/10.1002/cncr.11157. 415 23. Thompson MD, Monga SP. WNT/beta-catenin signaling in liver health and disease. 416 Hepatology 2007; 45:1298-305. https://doi.org/10.1002/hep.21651. 417 24. Gumbiner BM. Signal transduction of beta-catenin. Curr Opin Cell Biol 1995; 7:634-40. 418 https://doi.org/10.1016/0955-0674(95)80104-9. 419 25. Nei H, Saito T, Yamasaki H, Mizumoto H, Ito E, Kudo R. Nuclear localization of beta-420 catenin in normal and carcinogenic endometrium. Mol Carcinog 1999; 25:207-18. 421 https://doi.org/10.1002/(sici)1098-2744(199907)25:3<207::aid-mc7>3.0.co;2-4. 422 26. Morin PJ, Sparks AB, Korinek V, Barker N, Clevers H, Vogelstein B, Kinzler KW. 423 Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or 424 APC. Science 1997; 275:1787-90. https://doi.org/10.1126/science.275.5307.1787. 425 27. Watson JV, Curling OM, Munn CF, Hudson CN. Oncogene expression in ovarian cancer: a 426 pilot study of c-myc oncoprotein in serous papillary ovarian cancer. Gynecol Oncol 1987; 427 28:137-50. https://doi.org/10.1016/0090-8258(87)90207-1. 428 28. Odom LD, Barrett JM, Pantazis CG, Stoddard LD, McDonough PG. Immunocytochemical 429 study of ras and myc proto-oncogene polypeptide expression in the human menstrual cycle. 430 Am J Obstet Gynecol 1989; 161:1663-8. https://doi.org/10.1016/0002-9378(89)90946-0. 431 29. Bircan S, Ensari A, Ozturk S, Erdogan N, Dundar I, Ortac F. Immunohistochemical analysis 432 of c-myc, c-jun and estrogen receptor in normal, hyperplastic and neoplastic endometrium. 433 Pathol Oncol Res 2005; 11:32-9. https://doi.org/10.1007/bf03032403. 434 30. Geisler JP, Geisler HE, Manahan KJ, Miller GA, Wiemann MC, Zhou Z, Crabtree W. 435 Nuclear and cytoplasmic c-myc staining in endometrial carcinoma and their relationship to 436 survival. Int J Gynecol Cancer 2004;14:133-7. https://doi.org/10.1136/ijgc-00009577-437 200401000-00018. 438 439 Figure 1: A) Absence of signaling molecule i.e., legend (Wnt molecule) leads to formation of 440 ‘destruction complex’ that in turn creates a hyperphosphorylated β-catenin destined for 441 proteosomal degradation. Also depicted is E-cadherin binding to β-catenin forming adherens 442 junction. B) Wnt molecule binding to Frizzled (FZD)/LRP 5/6 receptors inactivates ‘destruction 443 complex’ and stabilizes hypophosphorylatedβ-catenin that enter nucleus to interact with 444 TCF/LEF family proteins to activate gene transcription. C) Dkk1 binds to LRP5/6 co-receptor 445 and blocks Wnt binding that ultimately results in β-catenin degradation and repression of gene 446 transcription. (Illustration is created by the authors). 447 448 449 Figure 2: Dkk1 immunopositivity. Proliferative endometrium (A, 400X magnification), 450 secretory endometrium (B, 400X magnification), endometrial atypical hyperplasia (C, 400X 451 magnification), and endometrioid carcinoma (D, 400X magnification). 452 453 Figure 3: E-cadherin immunopositivity. Proliferative endometrium (A, 400X magnification), 454 secretory endometrium (B, 400X magnification), endometrial atypical hyperplasia (C, 400X 455 magnification), and endometrioid carcinoma (D, 400X magnification). 456 457 458 Figure 4: β-catenin immunopositivity. Secretory endometrium (A, 400X magnification), 459 endometrial atypical hyperplasia (B, 100X magnification), endometrioid carcinoma (C, 400X 460 magnification), and nuclear positivity in endometrioid carcinoma (D, 400X magnification). 461 462 463 Figure 5: c-myc immunopositivity. Proliferative endometrium (A, 100X magnification), 464 secretory endometrium (B, 200X magnification), endometrial atypical hyperplasia (C, 200X 465 magnification), and endometrioid carcinoma (D, 400X magnification). 466 Table 1: Comparison of immunopositivity between grade I and grade II endometrioid carcinoma 467 IHC Grade (1 as Grade I, 2 as Grade II) No. of Cases Mean IRS±SD P value Dkk1 1 2 39 11 4.10±2.222 3.18±1.601 0.207 E-cadherin 1 2 39 11 2.92±1.645 2.82±1.662 0.853 β-catenin 1 2 39 11 3.31±1.360 3.64±1.502 0.492 c-myc 1 2 39 11 8.67±3.198 8.27±2.970 0.716 468