Anaplastic lymphoma kinase overexpression enhances an aggressive phenotypic characteristics of endometrial carcinoma

Anaplastic lymphoma kinase overexpression enhances an aggressive phenotypic characteristics of endometrial carcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Anaplastic lymphoma kinase overexpression enhances an aggressive phenotypic characteristics of endometrial carcinoma Ako Yokoi, Yusaku Nakamura, Miki Hashimura, Yasuko Oguri, Toshihide Matsumoto, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2203804/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Aug, 2023 Read the published version in BMC Cancer → Version 1 posted 9 You are reading this latest preprint version Abstract Background Although anaplastic lymphoma kinase (ALK) overexpression is overexpressed in several primary solid tumor types, its role in endometrial carcinoma (Em Ca) remains unclear. Methods We evaluated expression of ALK and its related molecules in clinical samples consisting of 168 Em Ca tissues. We also used Em Ca cell lines to evaluate the functional role of ALK. Results Cytoplasmic ALK immunoreactivity in the absence of chromosomal rearrangement was positively correlated with ALK mRNA expression, and was significantly higher in Grade (G) 3 Em Ca than in G1 or G2 tumors. ALK immunoreactivity was also significantly associated with expression of cancer stem cell (CSC)-related molecules (cytoplasmic CD133, ALDH1, Sox2) and neuroendocrine markers (CD56 and synaptophysin). Although the proliferative index was significantly higher in ALK-positive Em Ca when compared to ALK- negative malignancies, there was no association between ALK expression and other clinicopathological factors in this disease. In Em Ca cell lines, full-length ALK overexpression increased proliferation, decreased susceptibility to apoptosis, enhanced cancer stem cell features, and accelerated cell mobility, whereas these phenotypes were abrogated in ALK-knockdown cells. Finally, tumors with either wild-type ALK or high ALK mRNA expression were associated with a poorer prognosis when compared to Em Ca with either mutant ALK or low ALK mRNA expression. Conclusion Full-length ALK overexpression occurs in a subset of Em Ca, particularly in G3 tumors, and contributes to establishment and maintenance of aggressive phenotypic characteristics through modulation of several biological processes. ALK cancer stem cell epithelial-mesenchymal transition Neuroendocrine differentiation endometrial carcinoma Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The incidence of endometrial carcinoma (Em Ca), the most prevalent malignancy of the female genital tract in developing countries, is increasing [ 1 , 2 ]. Although most frequently observed in post-menopausal women, 20–25% of Em Ca are diagnosed before the menopause [ 1 , 2 ]. In Japan, the age-adjusted prevalence of Em Ca for women in 2014 was 16.0 per 100,000 and the overall rate has increased four-fold in the past 30 years, with a particularly rapid increase in women under 40 years old [ 3 , 4 ]. The most common risk factors associated with the development of Em Ca are unopposed estrogen exposure and obesity (type I tumors); a smaller subset of sporadic Em Ca is associated with aging and unique genetic and molecular changes that produce a more aggressive variant (type II tumors) [ 5 , 6 ]. Although most Em Ca patients are diagnosed at an early stage, 15–20% of tumors in these cases are advanced or recurrent diseases and are associated with a 5-year survival rate of 17% [ 8 ]. Thus, novel biomarkers and therapeutic targets for diagnosis or treatment of Em Ca are urgently required. The anaplastic lymphoma kinase (ALK) gene located on chromosome 2p23 belongs to the insulin receptor superfamily of receptor tyrosine kinases (RTK), and encodes a protein that is highly homologous to leukocyte tyrosine kinase (LTK). ALK consists of a large extracellular domain, a lipophilic transmembrane segment, and a cytoplasmic tyrosine kinase domain [ 9 – 13 ]. During normal embryogenesis, ALK is specifically expressed in the developing central and peripheral nervous system, where it may regulate the balance between proliferation and differentiation [ 14 – 16 ]. However, this kinase also plays a role in disease pathology. For example, chromosomal rearrangements in a subset of lymphomas and lung carcinomas give rise to several gene fusions that contain ALK [ 17 , 18 ]. Deregulated expression of full-length ALK has also been observed in some primary solid tumors including ovarian high-grade serous carcinoma and uterine carcinosarcoma [ 19 , 20 ]. Thus, aberrant ALK overexpression is closely associated with tumor development and progression of a variety of human malignancies. Here, we provide clear evidence that overexpression of full length ALK in the absence of chromosomal rearrangements occurs in a subset of Em Ca. Using both ALK overexpression and knockdown, we also demonstrate a role for ALK in the modulation of proliferation, apoptosis, and migration and induction of cancer stem cell (CSC) features in Em Ca cell lines and primary tumor samples. Methods Clinical cases Histological findings were reviewed in hysterectomy specimens of endometrioid-type Em Ca from the case records of Kitasato University Hospital between 2007 and 2020, according to the criteria of the 2014 World Health Organization classification [ 21 ]. Each case was also staged according to the 2009 International Federation of Gynecology and Obstetrics (FIGO) staging system and the TNM classification [ 22 ]. A total of 168 Em Ca cases, including 91 of grade (G)1, 40 of G2, and 37 of G3 were investigated. The mean age of the patients was 59.1 years (range, 31–92), and 113 were post-menopausal. Detailed clinicopathological data are provided in Supplementary Table S1. All tissues were routinely fixed in 10% formalin and processed for embedding in paraffin wax. Approval for this study was given by the Ethics Committee of the Kitasato University School of Medicine (B18-048). Antibodies and reagents Anti-ALK, anti-Rb phosphor-Ser807/811 (pRb), anti-CD56, anti-synaptophysin (Syn), and anti-vimentin, anti-Slug, anti-cleaved caspase-3, and anti-cleaved poly (ADP-ribose) polymerase 1 (PARP1) antibodies were purchased from Cell Signaling (Danvers, MA, USA). Anti-Sox7, anti-ZEB1, and anti-β-actin antibodies were obtained from Sigma-Aldrich Chemicals (St. Louis, MO, USA). Anti-Snail, anti-Nestin, and anti-Sox2 antibodies were from Abcam (Cambridge, MA, USA). Anti-Rb, anti-p27 kip1 , anti-N-cadherin, anti-BAX, anti-X-linked inhibitor of apoptosis (XIAP), and anti-aldehyde dehydrogenase (ALDH)1 antibodies were from BD Biosciences (San Jose, CA, USA). Anti-Ki-67, anti-p21 waf1 , anti-cyclin D1, anti-p53, anti-BCL2, and anti-CD44s antibodies were from Dako (Glostrup, Denmark). Anti-cyclin A2, anti-cyclin B1, and anti-CD133 antibodies were from Novocastra (Newcastle, UK), Santa Cruz Biotechnology (Santa Cruz, CA, USA), Miltenyi Biotechnology (Bergish Gladbach, Germany), respectively. Adriamycin (ADR: Catalog No. #D1515) was purchased from Sigma-Aldrich Chemicals. Immunohistochemistry (IHC) IHC was performed using a combination of the microwave oven heating and polymer immunocomplex (Envision, Dako) methods. For immunohistochemical detection of ALK, the ALK iAEP kit (Nichirei Biosciences, Tokyo, Japan) was applied. Lung carcinoma tissues with ALK overexpression due to a gene abnormality were used as positive controls, as described previously [ 19 , 20 ]. For evaluation of IHC findings, scoring of cytoplasmic immunoreactivity was performed on the basis of the percentage of immunopositive cells and the immunointensity with multiplication of the values of the two parameters as described previously [ 19 , 20 ]. Samples with ALK score with more than 1 were considered positive and those with scores less than 1 were considered negative on the basis of the average ALK score (means ± SDs = 0.69 ± 1.65). Nuclear Ki-67 immunoreactivity was also counted in at least 500 cells from five randomly selected fields and the labeling indices (LIs) were then calculated as a percentage. In addition, the number of cleaved PARP1-positive cells in five randomly selected fields was used to calculate the mean number of apoptotic cells per high-power field (HPF), as described previously [ 19 , 20 ]. RNAscope assay for ALK mRNA in situ hybridization Expression of ALK mRNA was analyzed using an RNAscope assay (Advanced Cell Diagnostics, Hayward, CA, USA) according to manufacturer’s instructions. The hybridization was performed with targeted probes: Hs-ALK (#311841), positive control probe (#2010684), and negative control probe (#310043) for 2 h at 40℃. ISH signal scores were classified into four levels, as follows: -, none; 1+, fewer than 10% positive cells; 2+, 10–30%; 3+, more than 30%. Plasmids and cell lines pcDNA3.1-full-length ALK and pSIREN-RetroQ-short hairpin (sh) ALK were used as described previously [ 19 , 20 ]. Eleven Em Ca cell lines (Ishikawa, Hec6, Hec50, Hec59, Hec88, Hec108, Hec116, Hec151, Hec155, Hec180, and Hec251), whcih we have established previously [ 23 , 24 ], were used. The full-length ALK expression plasmid or empty vector were transfected into Hec6 cells (which lack endogenous ALK expression) (Supplementary Figure S1A) and two stably overexpressing clones (H6-ALK#8 and H6-ALK#47) were established. ALK-knockdown lines (ALK-KD) were also generated using Hec59 cells (which have relatively high ALK expression) (Supplementary Figure S1B) and shRNA targeting the ALK gene (H59-shALK#11 and H59-shALK#33) as described previously [ 19 ]. Transfection Transfection was carried out using LipofectAMINE PLUS (Invitrogen) as described previously [ 19 , 20 ]. Reverse transcription (RT)-PCR cDNA was synthesized from 2 µg of total RNA. Amplification by RT-PCR was carried out in the exponential phase to allow comparisons between cDNA synthesized from identical reactions. Primers for the ALK and GAPDH genes were used as described previously [ 19 , 20 ]. Western blot assays Total cellular proteins were isolated using RIPA buffer [20 mM Tris-HCl (pH 7.2), 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate]. Aliquots of the proteins were resolved by SDS-PAGE, transferred to PVDF membranes, and probed with primary antibodies coupled to the ECL detection system (Amersham Pharmacia Biotechnology, Tokyo, Japan), as described previously [ 19 , 20 ].. Flow cytometry and Aldefluor assay Cells were fixed using 70% alcohol and stained with propidium iodide (Sigma) for cell cycle analysis. ALDH1 enzyme activity in viable cells was determined using a fluorogenic dye-based Aldefluor assay (Stem Cell Technologies, Grenoble, France) according to the manufacturer’s instructions. The prepared cells were analyzed by flow cytometry using BD FACS Calibur (BD Biosciences) and CellQuest Pro software version 3.3 (BD Biosciences), as described previously [ 19 , 20 ].. Spheroid assay Cells (x 10 3 ) were plated in low cell binding plates (Thermo Fisher Scientific, Yokohama, Japan) in Cancer Stem Cell Premium (ProMab Biotech, Richmond, CA). Uniform spheroids of at least 50 µm in diameter were counted approximately two weeks after plating, as described previously [ 19 , 20 ].. Wound healing assay Cells were seeded into 24-well tissue culture plates, and grown to reach 90–100% confluence. After a cell monolayer formed, a wound was scratched with a sterile 200-µl tip. The area of the wound was also analyzed using ImageJ software version 1.41. Closure of the wound was measured in pixels, and used as a measure of cell migration, as described previously [ 19 , 20 ].. FIuorescence in situ hybridization (FISH) For analysis of the ALK (2p23) locus, dual-color FISH studies were conducted on four HGSC cases with strong ALK immunopositivity using the Vysis LSI ALK break-apart rearrangement probe (Abbott Molecular, Abbott Park, IL, USA) according to the manufacturer’s instructions, as described previously [ 19 , 20 ].. Mutation analyses of the ALK gene Genomic DNA was extracted from Hec59 cells using a Wizard Genomic DNA Purification kit (Promega, Madison, WT, USA) according to the manufacturer’s instructions. Mutation analyses of exons 20, 23, 24, and 25 of the ALK gene were carried out as described previously [ 19 , 25 ]. Briefly, the PCR products were purified using a NucleoSpin Gel and PCR Clean-up (Macherey-Nagel, Düren, Germany) and bidirectional sequencing was performed using the BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, USA) on the ABI3130 genetic analyzer (Applied Biosystems). Sequencing Analysis software v5.4 (Applied Biosystems) along with a manual chromatogram review was used for sequence analysis. TCGA data analysis cBioportal ( http://www.cbioportal.org/ ) was used to extract The Cancer Genome Atlas (TCGA) HGSA ALK mutational data and expression data (RNA Seq V2 PSEM) associated with 506 Em Ca cases. ALK mRNA expression levels were subcategorized into ‘high’ and ‘low’ groups on the basis of the Z score (cutoff values were 0.5). Cancer Cell Line Encyclopedia (CCLE) data describing the ALK gene status and the relative expression of ALK mRNA in 28 Em Ca cell lines were also extracted from cBioPortal, as described previously [ 19 , 20 ].. Statistics Comparative data were analyzed using the Mann-Whitney U -test and Spearman’s correlation coefficient. Overall survival (OS) was calculated as the time between onset and death or the date of the last follow-up evaluation. Progression-free survival (PFS) was also examined from the onset of treatment until relapse, disease progression, or last follow-up evaluation. OS and PFS were estimated using the Kaplan-Meier method, and statistical comparisons were made using the log rank test. The cut-off for statistical significance was set as p < 0.05, as described previously [ 19 , 20 ].. Results ALK is overexpressed in a subset of Em Ca cases Representative images of IHC findings for ALK in Em Ca are illustrated in Fig. 1 A. Strong cytoplasmic ALK immunoreactivity was mainly observed in G3 Em Ca, in contrast to the weak membranous immunoreaction in G1 tumors. ALK immunopositivity was observed in 14 (15.4%) of 91 G1 Em Ca, 3 (7.5%) of 40 G2, and 17 (45.9%) of 37 G3; the difference between G3 scores and the other tumor grades was statistically significant (Supplementary Table S1). A similar finding was also observed with regard to average ALK scores (Fig. 1 A), whereas there were no associations between ALK expression and other clinicopathological factors in Em Ca (Supplementary Table S1). ALK immunoreactivity clearly overlapped with ALK mRNA expression, and the ALK IHC scores were significantly higher in the ALK ISH-high category when compared to the ALK ISH-low group (Fig. 1 B). In addition, FISH did not reveal ALK rearrangement or amplification in four Em Ca cases with strong ALK immunoreactivity (Fig. 1 C). Among eleven Em Ca cell lines, two (Hec59 and Hec251) had relatively high ALK mRNA expression (Supplementary Figure S1A), in line with CCLE data analyses (Supplementary Figure S1B). However, ALK protein was only detected in Hec59 cells (Supplementary Figure S1C). Neither Hec59 nor Hec251 cells had mutations in exons 20, 23, 23, 24, and 25 of the ALK gene (Supplementary Figure S1D); these data are consistent with CCLE data that show there is no association between ALK gene mutation and levels of ALK mRNA (Supplementary Table S2). Together, these findings suggest that cytoplasmic ALK expression without chromosomal rearrangement, along with the increased mRNA expression, is observed together with increased ALK mRNA expression in G3 Em Ca. ALK overexpression increased cell proliferation and decreased susceptibility of apoptosis in Em Ca cells To determine whether ALK played a functional role in Em Ca cells, we first established two independent Hec6 cell line clones stably overexpressing full-length ALK and two independent Hec59 cell line clones in which ALK expression was blocked by an ALK-specific shRNA. H6-ALK cells tended to proliferate rapidly than mock-transfected cells. This was consistent with the display of markers associated with cell cycle progression including increased G2/M fraction, increased Rb and pRb expression, and decreased p21 waf1 expression (Fig. 2 A). In contrast, H59-shALK cells tended to proliferate more slowly, and exhibited a reduced S phase fraction, although the expression of several cell cycle-related markers remained unchanged (Fig. 2 B). Compared with mock-transfected cells, ADR treatment of H6-ALK cells induced less cleaved caspase-3 expression and fewer apoptotic features, such as a less prominent sub-G1 FACS profile (Fig. 2 C and Supplementary S2A,B). The opposite effects were observed in ADR-treated H59-shALK cells (Fig. 2 D and Supplementary Figure S2C,D). Figure 3 A shows representative IHC images for Ki-67 and cleaved PARP1 in ALK-high and ALK-low G3 Em Ca cases. There was a positive correlation between the two markers in Em Ca (Table 1). Both Ki-67 and cleaved PARP1 immunoreactivities were significantly higher in G3 Em Ca than in G1/G2 tumors. Average Ki-67 LI values were also significantly higher in ALK-positive tumors when compared with their ALK-negative counterparts (Fig. 3 B), and ALK score was also positively and significantly correlated with Ki-67 LI (Table 1). However, there were no correlations between ALK levels and cleaved PARP1 score (Fig. 3 C and Table 1). These findings suggest that ALK overexpression increases proliferation and reduces susceptibility to apoptosis in Em Ca cells. ALK overexpression enhances CSC features and migration in Em Ca cells ALK induces CSC features through activation of the epithelial-mesenchymal transition (EMT) in ovarian high-grade serous carcinomas and uterine carcinosarcoma [ 19 , 20 ]. We therefore examined whether the association between ALK expression and CSC properties was also feature of Em Ca. Indeed, H6-ALK cells exhibited increased expression of several CSC and EMT markers including CD133, CD44s, Nestin, Sox2, Snail, Slug, and ZEB1 when compared to mock cells (Fig. 4 A). The Aldefluor assay also revealed a significant ALDH1 high population in the H6-ALK cells (Fig. 4 B), in line with a significant increase in the number of well-defined, round spheroids that were over 50 mm in diameter (Fig. 4 C). In contrast, levels of these markers were significantly lower in H59-shALK cells (Fig. 4 D,E,F). We then examined whether ALK expression modulates cell motility using the wound healing assay. H6-ALK cells refilled wounded empty spaces more rapidly, and exhibited significantly increased migration capacity (Fig. 5 A). Conversely, H59-shALK cells were less able to refill the empty wound spaces, and had lower migration rates than mock cells (Fig. 5 B). Since ALK overexpression is associated with neuroendocrine (NE) differentiation and TP53 gene status in ovarian high-grade serous carcinomas and uterine carcinosarcoma [ 19 , 20 ], we investigated NE markers and p53 staining in Em Ca (Fig. 6 ). Distinct cytoplasmic and/or membranous immunostaining for CD56, Syn, CD133, and ALDH1, as well as nuclear immunoreactivity for p53, Sox2, and Sox7 were observed in Em Ca cells. Average IHC scores for CD56, Syn, p53, cytoplasmic (Cyt)-CD133, ALDH1, and Sox2, as well as ALK, were significantly higher in G3 Em Cas as compared to those of G1 or G2 tumors, but this differential pattern was not evident for membranous CD133 or Sox7 (Fig. 6 B). In addition, ALK score was positively correlated with CD56, Syn, Cyt-CD133, ALDH1, Sox2, Sox7, and p53 scores. Both Sox2 and Cyt-CD133 scores were also positively associated with a subset of NE and CSC markers (Table 1). These findings suggest that ALK overexpression enhances CSC properties, migration capability, and NE features in Em Ca cells. Correlation between ALK status and survival in Em Ca Kaplan-Meier curves showed that Em Ca patients with high ALK mRNA expression and wild-type ALK gene status had poorer OS and PFS when compared to patients with low ALK mRNA expression and mutant ALK (Supplementary Figure S3). Discussion The present study clearly provides evidence of a positive correlation between ALK mRNA and protein in Em Ca tissues. Furthermore, we found no evidence of ALK chromosomal rearrangements or mutations in both Em Ca clinical samples or cell lines. We previously showed that full length ALK overexpression was under the transcriptional control of SoxB1 (Sox2 and Sox3) and SoxF (Sox7 and Sox17) in ovarian high-grade serous carcinoma [ 19 ], suggesting the existence of a positive feedback loop between ALK and Sox factors. We also found that ALK promoter activity was suppressed by wild-type p53, but not mutant p53 [ 19 ]. In general, a high p53 score (60–100% immunopositive cells) is sufficient to identify a p53 mutation in 94% of cases [ 26 ]. Given our present finding that ALK expression was positively correlated with Sox2, Sox7, and p53 scores in Em Ca tissues, we suggest that transcriptional upregulation of ALK may also occur in Em Ca via transcriptional activators including Sox factors and p53. We also found that ALK overexpression was significantly higher in G3 Em Ca when compared to G1/G2 tumors. ALK was predominantly cytoplasmic in G3 tumors, and membrane localized in G1/G2 tumors. Moreover, ALK expression was associated with increased proliferation but not with levels of cleaved PARP1 in Em Ca cells. This is consistent with evidence that cytoplasmic localization of the ALK tyrosine kinase domain promotes proliferation, whereas membrane attachment is critical for initiation of neurite outgrowth and proliferation arrest [ 27 ]. Interestingly, stable ALK overexpression in Em Ca cell lines significantly increased proliferation, reduced susceptibility to apoptosis, and these effects were abrogated in ALK-knockdown cells. Given that loss of ALK inhibits the growth of breast carcinoma cell lines [ 28 ], we suggest that cytoplasmic ALK modulates cell survival and proliferation. This is probably mediated via activation of ALK-related signaling pathways including phosphatidylinositol 3-kinase-AKT, Janus Kinase-STAT, and mitogen-activated protein kinase pathways, which contribute to regulation of cell cycle progression and apoptotic features [ 29 ]. An important finding of this study is that ALK overexpression enhanced CSC properties; this is consistent with the upregulation of several CSC markers, including Cyt-CD133, ALDH1, and Sox2 in Em Ca tissues. Moreover, ALK overexpression significantly enhanced migration capability, whereas ALK knockdown abrogates this effect. In glioblastoma, CSCs are more invasive than non-CSCs [ 30 ]. Taking these results together, we infer that the increased migration capability of CSC-like cells may contribute to the aggressive features of ALK-overexpressing Em Ca cells. Finally, overexpression of ALK, as well as Sox2, was also clearly associated with expression of NE markers in G3 Em Ca cells. Similar findings were also observed in ovarian high-grade serous carcinoma [ 19 ]. In general, the prognosis of NE carcinoma is poor, as more than 80% of patients present with metastatic disease at diagnosis, and there are few effective therapies for this cancer type [ 31 ]. Given that Sox2 blocks the differentiation of the neural progenitor population [ 32 ], we suggest that the combined activities of ALK and Sox2 induce an NE-like phenotype in Em Ca cells, and that this contributes to the poor OS and PFS in Em Ca. Our hypothesis was supported by analysis of TCGA data, which showed that high ALK mRNA expression and wild-type ALK status were associated with poorer prognosis in Em Ca cases. Conclusion Our overall conceptual framework for the role of ALK in aggressive Em Ca is summarized in Fig. 7 . Full-length ALK overexpression occurs in a subset of Em Ca, particularly in G3 tumors, and contributes to establishment and maintenance of aggressive phenotypic characteristics through modulation of several biological processes. Abbreviations ALK, anaplastic lymphoma kinase; Em Ca, endometrial carcinoma; CSC, cancer stem cell; EMT, epithelial-mesenchymal transition; IHC, immunohistochemistry; NE, neuroendocrine Declarations Acknowledgements No applicable Authors’ contributions statement AY, YN, and MS carried out the majority of the experiments, analyzed the data, and wrote the manuscript. They were helped by MH, YO, TM, MN, YI, TI, KO, YH, and NF. All authors reviewed and approved the final manuscript. Funding This study was supported by a grant from JSPS KAKENHI Grant Number 21K20781 Availability of data and materials The data sets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate This study was approved by the Kitasato University Medical Ethics Committee (B20-81) and was conducted according to the Helsinki Declaration. The requirement for informed consent was waived by the Kitasato University Hospital, as this was a retrospective study. Consent for publication Not applicable Competing interests All authors declared no competing interests. Author details 1 Department of Pathology, Kitasato University School of Medicine, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0374, Japan. 2 Department of Pathology, Kitasato University School of Allied Health Science, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0374, Japan. References Ryan AJ, Susil B, Jobling TW, Oehler MK. Endometrial cancer. Cell Tissue Res. 2005;332:53–61. Kasoha M, Dernektsi C, Seibold A, Bohle RM, Takacs Z, Ioan-lulian I, Solomayer E-F, Juhasz-Boss I. Crosstalk of estrogen receptors and Wnt/β-catenin signaling in endometrial cancer. J Cancer Res Clin Oncol. 2020;146:315–27. Ushijima K. Current status of gynecologic cancer in Japan. J Gynecol Oncol. 2009;20:67–71. Yamagami W, Mikami M, Nagase S, Tabata T, Kobayashi Y, Kaneuchi M, et al. Japan society of gynecologic oncology 2018 guidelines for treatment of uterine body neoplasms. J Gynecol Oncol. 2020;31:e18. Cragun JM, Havrilesky LJ, Calingaert B, Synan I, Secord AA, Soper JT, et al. Retrospective analysis of selective lymphoadenectomy in aapparent early-stage endometrial cancer. J Clin Oncol. 2005;23:3668–75. ASTEC study group. Kitchener H, Swart AM, Qian Q, Amos C, Parmar MKB. Efficacy of systematic pelvic lymphoadenectomy in endometrial cancer (MRC ASTEC trial): a randomized study. Lancet. 2009;373:125–36. Benedetti Panici P, Basile S, Maneschi F, Alberto Lissoni A, Signorelli M, Scambia G, et al. Systematic pelvic lymphoadenectomy vs. no lymphadenectomy in early-stage endometrial carcinoma: randomized clinical trial. J Natl Cancer Inst. 2008;100:1707–16. Siegel RL, Miller KD, Jemal A. Cancer statistics. CA Cancer J Clin. 2016;66:7–30. Iwahara T, Fujimoto J, Wen D, Cupples R, Bucay N, Arakawa T, et al. Molecular characterization of ALK, a receptor tyrosine kinase expressed specifically in the nervous system. Oncogene. 1997;14:439–49. Morris SW, Naeve C, Mathew P, James PL, Kirstein MN, Cui X, et al. ALK, the chromosome 2 gene locus altered by the t(2;5) in non-Hodgkin’s lymphoma, encodes a novel neural receptor tyrosine kinase that is highly related to leukocyte tyrosine kinase (LTK). Oncogene. 1997;14:2175–88. Ben-Neriah Y, Bauskin AR. Leukocytes express a novel gene encoding a putative transmembrane protein-kinase devoid of an extracellular domain. Nature. 1988;333:672–6. Maru Y, Hirai H, Takaku F. Human Itk: gene structure and preferential expression in human leukemic cells. Oncogene Res. 1990;5:199–204. Bernards A, de la Monte SM. The Itk receptor tyrosine kinase is expressed in pre-B lymphocytes and cerebral neurons and uses a non-AUG translational initiator. EMBO J. 1990;2:2279–87. Cheung NK, Dyer MA. Neuroblastoma: developmental biology, cancer genomics and immunotherapy. Nat Rev Cancer. 2013;13:397–411. Reiff T, Huber L, Kramer M, Delattre O, Janoueix-Lerosey I, Rohrer H. Midkine and Alk signaling in sympathetic neuron proliferation and neuroblastoma predisposition. Development. 2011;13:685–700. Hallberg B, Palmer RH. Mechanistic insight into ALK receptor tyrosine kinase in human cancer biology. Nat Rev Cancer. 2013;13:685–700. Morris SW, Naeve C, Mathew P, James PL, Kirstein MN, Cui X, et al. ALK, the chromosome 2 gene locus altered by the t(2;5) in non-Hodgkin’s lymphoma, encodes a novel neural receptor tyrosine kinase that is highly related to leukocyte tyrosine kinase (LTK). Oncogene. 1997;14:2175–88. Takeuchi K, Soda M, Togashi Y, Suzuki R, Sakata S, Hatano S, et al. RET, ROS1 and ALK fusions in lung cancer. Nat Med. 2012;18:378–81. Matsumoto T, Oda Y, Hasegawa Y, Hashimura M, Oguri Y, Inoue H, et al. Anaplastic lymphoma Kinase overexpression is associated with aggressive phenotypic characteristics of ovarian high-grade serous carcinoma. Am J Pathol. 191:1837–1850. Inoue H, Hashimura M, Akiya M, Chiba R, Saegusa M. Functional role of ALK-related signal cascades on modulation of epithelial-mesenchymal transition and apoptosis in uterine carcinosarcoma. Mol Cancer. 2017;16:37. Zaino R, Carinelli SG, Ellenson LH, Eng C, Katabuchi H, Konishi I, et al Tumours of the uterine corpus. In: Kurman RJ, Carcangiu ML, Herrington CS, Young RH, editors. WHO classification of tumours of female reproductive organs. Lyon: IARC; 2014. P.121 – 54. Creasman W. Revised FIGO staging for carcinoma of the endometrium. Int J Gynaecol Obstet. 2009;105:109. Kuramoto H, Hamano M, Imai M, Fijisawa T, Kamata Y, Arai T, et al. Hec-1 cells: establishment of an in vitro experimental system in endometrial carcinoma. Cell and molecular biology of endometrial carcinoma. Edited by Kuramoto H, Nishida M. Tokyo, Springer-Verlag, 2003, p. 3–34. Nishida M. Ishikawa cells: opening of in vitro hormone research on endometrial carcinoma. Cell and molecular biology of endometrial carcinoma. Edited by Kuramoto H, Nishida M. Tokyo, Springer-Verlag, 2003, p. 35–60. Chiba R, Akiya M, Hashimura M, Oguri Y, Inukai M, Hara A, et al. ALK signaling cascade confers multiple advantages to glioblastoma cells through neovascularization and cell proliferation. PLoS ONE. 2017;12:e0183526. Yemelyanova A, Vang R, Kshirsagar M, Lu D, Marks MA, Shih leM, Kurman RJ. Immunohistochemical staining patterns of p53 can serve as a surrogate marker for TP53 mutations in ovarian carcinomas: an immunohistochemical and nucleotide sequencing analysis. Mod Pathol. 2011;24:1248–53. Gouzi JY, Moog-Lutz C, Vigny M, Brunet-de Carvalho N. Role of the subcellular localization of ALK tyrosine kinase domain in neuronal differentiation of PC12 cells. J Cell Sci. 2005;118:5811–23. Siraj AK, Beg S, Jehan Z, Prabhakaran S, Ahmed M, R.Hussain A, et al. ALK alteration is a frequent event in aggressive breast cancers. Breast Cancer Res. 2015;17:127. Hallberg B, Palmer RH. The role of the ALK receptor in cancer biology. Ann Oncol. 2016;27:iii4–15. Volovetz J, Berezovsky AD, Alban T, Chen Y, Lauko A, Aranjuez GF, et al. Identifying conserved molecular targets required for cell migration of glioblastoma cancer stem cells. Cell Death Dis. 2020;11:152. Freis P, Graillot E, Rousset P, Hervieu V, Chardon L, Lombard-Bohas C, et al. Prognostic factors in neuroendocrine carcinoma: biological markers are more useful than histomorphological markers. Sci Rep. 2017;7:40609. Kiefer JC. Back to basics. Sox genes. Dev Dyn. 2007;236:2356–66. Tables Tables 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.xlsx SupFigureS1.tif SupFigureS2.tif SupFigureS3.tif SupTableS1.xlsx SupTableS2.xlsx Supplementarysection.docx Cite Share Download PDF Status: Published Journal Publication published 17 Aug, 2023 Read the published version in BMC Cancer → Version 1 posted Editorial decision: Major revision 25 Apr, 2023 Reviews received at journal 11 Mar, 2023 Reviewers agreed at journal 03 Mar, 2023 Reviewers agreed at journal 03 Mar, 2023 Reviewers invited by journal 27 Feb, 2023 Editor assigned by journal 17 Feb, 2023 Editor invited by journal 01 Dec, 2022 Submission checks completed at journal 01 Dec, 2022 First submitted to journal 25 Oct, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2203804","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":156754493,"identity":"ba126981-1839-4f99-bb60-d4275523e09a","order_by":0,"name":"Ako Yokoi","email":"","orcid":"","institution":"Kitasato University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ako","middleName":"","lastName":"Yokoi","suffix":""},{"id":156754494,"identity":"767f7b16-c85d-4424-ad47-486579b9a5b6","order_by":1,"name":"Yusaku Nakamura","email":"","orcid":"","institution":"Kitasato University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yusaku","middleName":"","lastName":"Nakamura","suffix":""},{"id":156754495,"identity":"1cfa4ed8-f773-42ee-87c3-9c653fa01df2","order_by":2,"name":"Miki Hashimura","email":"","orcid":"","institution":"Kitasato University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Miki","middleName":"","lastName":"Hashimura","suffix":""},{"id":156754496,"identity":"3628fcb6-406e-4356-ad4e-c97f239f4893","order_by":3,"name":"Yasuko Oguri","email":"","orcid":"","institution":"Kitasato University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yasuko","middleName":"","lastName":"Oguri","suffix":""},{"id":156754497,"identity":"8b3ee49f-494e-4580-bbda-ae1469fa5ed8","order_by":4,"name":"Toshihide Matsumoto","email":"","orcid":"","institution":"Kitasato University School of Allied Health Science","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Toshihide","middleName":"","lastName":"Matsumoto","suffix":""},{"id":156754498,"identity":"de83a045-c670-4d1f-98ae-b195f063436f","order_by":5,"name":"Mayu Nakagawa","email":"","orcid":"","institution":"Kitasato University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mayu","middleName":"","lastName":"Nakagawa","suffix":""},{"id":156754499,"identity":"412c496d-1c32-401e-a974-4c9b988439c6","order_by":6,"name":"Yu Ishibashi","email":"","orcid":"","institution":"Kitasato University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Ishibashi","suffix":""},{"id":156754500,"identity":"49c08612-54af-4222-916b-4ccf34173f02","order_by":7,"name":"Takashi Ito","email":"","orcid":"","institution":"Kitasato University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Takashi","middleName":"","lastName":"Ito","suffix":""},{"id":156754501,"identity":"3741d243-f04d-4601-9161-f3e66de55332","order_by":8,"name":"Kensuke Ohhigata","email":"","orcid":"","institution":"Kitasato University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kensuke","middleName":"","lastName":"Ohhigata","suffix":""},{"id":156754502,"identity":"592872f1-b9eb-43cf-8a1f-9e4243d5dd75","order_by":9,"name":"Youhei Harada","email":"","orcid":"","institution":"Kitasato University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Youhei","middleName":"","lastName":"Harada","suffix":""},{"id":156754503,"identity":"28fabeca-954d-401d-91c2-79ec47f69e26","order_by":10,"name":"Naomi Fukagawa","email":"","orcid":"","institution":"Kitasato University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Naomi","middleName":"","lastName":"Fukagawa","suffix":""},{"id":156754504,"identity":"8bfb6727-7784-422f-b767-f90d7b8cea82","order_by":11,"name":"Makoto Saegusa","email":"data:image/png;base64,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","orcid":"","institution":"Kitasato University School of Medicine","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Makoto","middleName":"","lastName":"Saegusa","suffix":""}],"badges":[],"createdAt":"2022-10-25 21:44:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2203804/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2203804/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12885-023-11144-2","type":"published","date":"2023-08-17T22:02:52+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":29914198,"identity":"5ce81e45-0260-4f71-9b0b-52dad2a6d8b3","added_by":"auto","created_at":"2022-12-05 16:06:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":12634470,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eALK mRNA and protein overexpression without gene alterations in Em Ca. \u003c/strong\u003e(A) Left: HE and IHC staining for ALK in G1 and G3 Em Ca. Note the strong cytoplasmic ALK immunoreactivity in G3 Em Ca, in contrast to the membranous immunoreactivity in G1 tumors. Closed boxes are magnified in the insets. Original magnification, x100 and x400 (inset). Right: IHC scores for ALK in Em Ca. The data shown are means ± SDs. (B) Left: HE, RNAscope, and IHC for ALK. Note the overlapping between ALK mRNA and protein expression in Em Ca tissues. Closed boxes are magnified in the insets. Original magnification, x100 and x400 (inset). Right: relationship between IHC scores and ISH signals for ALK in Em Ca. The data shown are means ± SDs. (C) FISH analysis of four Em Ca cases with high ALK scores. The interphase nuclei of both cases confirm the absence of \u003cem\u003eALK\u003c/em\u003erearrangement, in which the red and green signals remain fused.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-2203804/v1/2988d8e564aa7a40d6404f0d.png"},{"id":29914206,"identity":"164d0e6b-9127-45a1-a901-3239be3fab83","added_by":"auto","created_at":"2022-12-05 16:06:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4336659,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRelationship between ALK expression, proliferation, and apoptosis in Em Ca.\u003c/strong\u003e (A, B) Left upper: two independent H6-ALK (A), H59-shALK (B), and mock cell lines were seeded at low density. Cell numbers are presented as means ± SDs. P0, P3, P6, and P9 are 0, 3, 6, and 9 days after seeding, respectively. Left lower: FACS analysis of H6-ALK (A), H59-shALK (B), and mock cells. Right: western blot analysis for the indicated proteins in total lysates from H6-ALK (A), H59-shALK (B), and mock cells. (C,D) Left: FACS analysis of H6-ALK (C), H59-shALK (D), and mock cells treated with 1 mg/mL Adriamycin (ADR) for the time shown. † symbols indicate the sub-G1 fraction. Right: western blot analysis for the indicated proteins in total lysates from H6-ALK (C), H59-shALK (D), and mock cells treated with 1 mg/mL ADR for the time shown.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-2203804/v1/ecb31a8757264f59e423f315.png"},{"id":29915249,"identity":"01b98559-eede-4965-bb36-519aa25e780a","added_by":"auto","created_at":"2022-12-05 16:22:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":9399767,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRelationship between ALK expression, cell proliferation, and apoptosis in Em Ca. \u003c/strong\u003e(A) HE and IHC staining for ALK, Ki-67, and cleaved PARP1 in G3 Em Ca. Closed boxes are magnified in the insets. Original magnification, x100 and x400 (inset). (B,C) Left: Ki-67 LIs (B) and number of cleaved PARP1-positive cells (C) between G1, G2, and G3 Em Ca. Right: Ki-67 LIs (B) and number of cleaved PARP1-positive cells (C) between ALK-positive and -negative Em Ca. The data shown are means ± SDs.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-2203804/v1/8aa239bba8ed5d0524f72809.png"},{"id":29914211,"identity":"441b22b3-757f-4683-b892-755df5c42d17","added_by":"auto","created_at":"2022-12-05 16:06:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4847729,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges in CSC properties in H6-ALK and H59-shALK cells\u003c/strong\u003e. (A, D) Western blot analysis for the indicated proteins in total lysates from H6-ALK (A), H59-shALK (D), and mock cells (Mo). (B, E) Aldefluor analysis of H6-ALK (B), H59-shALK (E), and mock cells. Cells with no ALDH1 activity are located in the area to the far left of each plot, and the positive cells are within the black gate (R1). The percentage of live single cell populations contained in each gate is shown. DEAB, dimethylaminobenzaldehyde. (C, F) Upper: phase-contrast photograms of spheroids derived from H6-ALK (C), H59-shALK (F), and mock cells following 2 weeks of growth. Scale bars = 50 mm. Lower: numbers of spheroids are presented as means ± SDs. Mo, mock controls.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-2203804/v1/32ab315d7a4a85446d8c6c74.png"},{"id":29914209,"identity":"ab1314ae-82cc-410f-940d-54b7a7f3a2e1","added_by":"auto","created_at":"2022-12-05 16:06:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":11278026,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChange in cell migration potential of H6-ALK and H59-shALK cells.\u003c/strong\u003e (A, B) Left upper: wound healing assay with H6-ALK (A), H59-shALK (B), and mock cells. A scratch was made in the middle of a layer of confluent cells, and phase contrast images were taken over the indicated time period. The red dotted lines indicate the borders between confluent cell layers and wound areas. Left lower: the values of wound areas were calculated using NIH ImageJ software with those at 0 h set as 1. The fold wound areas are presented as means ± SDs. Mo, mock controls. The experiment was performed in duplicate. Right upper: H6-ALK, H59-shALK and mock cells were seeded in a 24-well transwell plates and incubated for 24 h in medium without serum. Cells were stained with HE and counted using a light microscope. Right lower: the numbers of migrated cells are presented as means ± SDs. Mo, mock controls. The experiment was performed in duplicate.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-2203804/v1/48bb585503f9a8f957601f74.png"},{"id":29914199,"identity":"70842c2b-86c3-46a1-9945-db9e9eb16f8c","added_by":"auto","created_at":"2022-12-05 16:06:38","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":13285960,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIHC in serial sections of Em Ca samples.\u003c/strong\u003e (A) Staining by HE and IHC for the indicated proteins in G3 Em Ca. Closed boxes are magnified in the insets. Original magnification, x100 and x400 (inset). (B) IHC scores for the indicated proteins in Em Ca. The data shown are means ± SDs.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-2203804/v1/3dfec765a2229ceba8b57a08.png"},{"id":29914202,"identity":"313f6e42-b068-4e5d-b603-9c0c0a338a56","added_by":"auto","created_at":"2022-12-05 16:06:38","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":282177,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic representation of the functional roles of ALK in an aggressive phenotype of Em Ca.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-2203804/v1/c3880ffc86f69e2eb4db4c5f.png"},{"id":44735780,"identity":"0b20c56f-fb12-47f9-93ea-35e79a03aab4","added_by":"auto","created_at":"2023-10-16 22:27:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3752067,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2203804/v1/962c6c4d-107a-47d9-bf03-830630805bdf.pdf"},{"id":29915047,"identity":"5c90b085-54f5-4900-b926-2eca2708d707","added_by":"auto","created_at":"2022-12-05 16:14:38","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":11679,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-2203804/v1/71c383dc9fba3b0db2eb98bd.xlsx"},{"id":29914200,"identity":"6732dd1c-e8ae-462c-8132-9ba29a41c478","added_by":"auto","created_at":"2022-12-05 16:06:38","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":4611980,"visible":true,"origin":"","legend":"","description":"","filename":"SupFigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-2203804/v1/c2ed10835ba711812b7a7985.tif"},{"id":29915050,"identity":"7467fe54-d967-4a6d-a3cd-991bdbdee9b8","added_by":"auto","created_at":"2022-12-05 16:14:38","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":8260092,"visible":true,"origin":"","legend":"","description":"","filename":"SupFigureS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-2203804/v1/45417e7817829dbe5eb545e8.tif"},{"id":29914203,"identity":"51d05b62-86fb-498b-9b30-c8a55d4278eb","added_by":"auto","created_at":"2022-12-05 16:06:38","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1748280,"visible":true,"origin":"","legend":"","description":"","filename":"SupFigureS3.tif","url":"https://assets-eu.researchsquare.com/files/rs-2203804/v1/ff846d08d715bbc46e9757b8.tif"},{"id":29915049,"identity":"ebe73c52-6d54-455b-b78c-f9cd42788a8d","added_by":"auto","created_at":"2022-12-05 16:14:38","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":10994,"visible":true,"origin":"","legend":"","description":"","filename":"SupTableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-2203804/v1/ef0483b1b7d2c34e49413e70.xlsx"},{"id":29914205,"identity":"3e6b9b33-2cda-4892-b7c1-8b6597f3b090","added_by":"auto","created_at":"2022-12-05 16:06:38","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":11103,"visible":true,"origin":"","legend":"","description":"","filename":"SupTableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-2203804/v1/5a7bf687b6650cd411dc24ea.xlsx"},{"id":29914207,"identity":"f95687e8-24cf-433e-8693-7deae70341be","added_by":"auto","created_at":"2022-12-05 16:06:38","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":20752,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarysection.docx","url":"https://assets-eu.researchsquare.com/files/rs-2203804/v1/87fa907e107bf388125d032e.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Anaplastic lymphoma kinase overexpression enhances an aggressive phenotypic characteristics of endometrial carcinoma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe incidence of endometrial carcinoma (Em Ca), the most prevalent malignancy of the female genital tract in developing countries, is increasing [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Although most frequently observed in post-menopausal women, 20\u0026ndash;25% of Em Ca are diagnosed before the menopause [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In Japan, the age-adjusted prevalence of Em Ca for women in 2014 was 16.0 per 100,000 and the overall rate has increased four-fold in the past 30 years, with a particularly rapid increase in women under 40 years old [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The most common risk factors associated with the development of Em Ca are unopposed estrogen exposure and obesity (type I tumors); a smaller subset of sporadic Em Ca is associated with aging and unique genetic and molecular changes that produce a more aggressive variant (type II tumors) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Although most Em Ca patients are diagnosed at an early stage, 15\u0026ndash;20% of tumors in these cases are advanced or recurrent diseases and are associated with a 5-year survival rate of 17% [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Thus, novel biomarkers and therapeutic targets for diagnosis or treatment of Em Ca are urgently required.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eanaplastic lymphoma kinase (ALK)\u003c/em\u003e gene located on chromosome 2p23 belongs to the insulin receptor superfamily of receptor tyrosine kinases (RTK), and encodes a protein that is highly homologous to leukocyte tyrosine kinase (LTK). ALK consists of a large extracellular domain, a lipophilic transmembrane segment, and a cytoplasmic tyrosine kinase domain [\u003cspan additionalcitationids=\"CR10 CR11 CR12\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. During normal embryogenesis, ALK is specifically expressed in the developing central and peripheral nervous system, where it may regulate the balance between proliferation and differentiation [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, this kinase also plays a role in disease pathology. For example, chromosomal rearrangements in a subset of lymphomas and lung carcinomas give rise to several gene fusions that contain ALK [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Deregulated expression of full-length ALK has also been observed in some primary solid tumors including ovarian high-grade serous carcinoma and uterine carcinosarcoma [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Thus, aberrant ALK overexpression is closely associated with tumor development and progression of a variety of human malignancies.\u003c/p\u003e \u003cp\u003eHere, we provide clear evidence that overexpression of full length ALK in the absence of chromosomal rearrangements occurs in a subset of Em Ca. Using both ALK overexpression and knockdown, we also demonstrate a role for ALK in the modulation of proliferation, apoptosis, and migration and induction of cancer stem cell (CSC) features in Em Ca cell lines and primary tumor samples.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eClinical cases\u003c/p\u003e \u003cp\u003eHistological findings were reviewed in hysterectomy specimens of endometrioid-type Em Ca from the case records of Kitasato University Hospital between 2007 and 2020, according to the criteria of the 2014 World Health Organization classification [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Each case was also staged according to the 2009 International Federation of Gynecology and Obstetrics (FIGO) staging system and the TNM classification [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. A total of 168 Em Ca cases, including 91 of grade (G)1, 40 of G2, and 37 of G3 were investigated. The mean age of the patients was 59.1 years (range, 31\u0026ndash;92), and 113 were post-menopausal. Detailed clinicopathological data are provided in Supplementary Table S1. All tissues were routinely fixed in 10% formalin and processed for embedding in paraffin wax. Approval for this study was given by the Ethics Committee of the Kitasato University School of Medicine (B18-048).\u003c/p\u003e \u003cp\u003eAntibodies and reagents\u003c/p\u003e \u003cp\u003eAnti-ALK, anti-Rb phosphor-Ser807/811 (pRb), anti-CD56, anti-synaptophysin (Syn), and anti-vimentin, anti-Slug, anti-cleaved caspase-3, and anti-cleaved poly (ADP-ribose) polymerase 1 (PARP1) antibodies were purchased from Cell Signaling (Danvers, MA, USA). Anti-Sox7, anti-ZEB1, and anti-β-actin antibodies were obtained from Sigma-Aldrich Chemicals (St. Louis, MO, USA). Anti-Snail, anti-Nestin, and anti-Sox2 antibodies were from Abcam (Cambridge, MA, USA). Anti-Rb, anti-p27\u003csup\u003ekip1\u003c/sup\u003e, anti-N-cadherin, anti-BAX, anti-X-linked inhibitor of apoptosis (XIAP), and anti-aldehyde dehydrogenase (ALDH)1 antibodies were from BD Biosciences (San Jose, CA, USA). Anti-Ki-67, anti-p21\u003csup\u003ewaf1\u003c/sup\u003e, anti-cyclin D1, anti-p53, anti-BCL2, and anti-CD44s antibodies were from Dako (Glostrup, Denmark). Anti-cyclin A2, anti-cyclin B1, and anti-CD133 antibodies were from Novocastra (Newcastle, UK), Santa Cruz Biotechnology (Santa Cruz, CA, USA), Miltenyi Biotechnology (Bergish Gladbach, Germany), respectively. Adriamycin (ADR: Catalog No. #D1515) was purchased from Sigma-Aldrich Chemicals.\u003c/p\u003e \u003cp\u003eImmunohistochemistry (IHC)\u003c/p\u003e \u003cp\u003eIHC was performed using a combination of the microwave oven heating and polymer immunocomplex (Envision, Dako) methods. For immunohistochemical detection of ALK, the ALK iAEP kit (Nichirei Biosciences, Tokyo, Japan) was applied. Lung carcinoma tissues with ALK overexpression due to a gene abnormality were used as positive controls, as described previously [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor evaluation of IHC findings, scoring of cytoplasmic immunoreactivity was performed on the basis of the percentage of immunopositive cells and the immunointensity with multiplication of the values of the two parameters as described previously [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Samples with ALK score with more than 1 were considered positive and those with scores less than 1 were considered negative on the basis of the average ALK score (means\u0026thinsp;\u0026plusmn;\u0026thinsp;SDs\u0026thinsp;=\u0026thinsp;0.69\u0026thinsp;\u0026plusmn;\u0026thinsp;1.65). Nuclear Ki-67 immunoreactivity was also counted in at least 500 cells from five randomly selected fields and the labeling indices (LIs) were then calculated as a percentage. In addition, the number of cleaved PARP1-positive cells in five randomly selected fields was used to calculate the mean number of apoptotic cells per high-power field (HPF), as described previously [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRNAscope assay for ALK mRNA \u003cem\u003ein situ\u003c/em\u003e hybridization\u003c/p\u003e \u003cp\u003eExpression of ALK mRNA was analyzed using an RNAscope assay (Advanced Cell Diagnostics, Hayward, CA, USA) according to manufacturer\u0026rsquo;s instructions. The hybridization was performed with targeted probes: Hs-ALK (#311841), positive control probe (#2010684), and negative control probe (#310043) for 2 h at 40℃. ISH signal scores were classified into four levels, as follows: -, none; 1+, fewer than 10% positive cells; 2+, 10\u0026ndash;30%; 3+, more than 30%.\u003c/p\u003e \u003cp\u003ePlasmids and cell lines\u003c/p\u003e \u003cp\u003epcDNA3.1-full-length ALK and pSIREN-RetroQ-short hairpin (sh) ALK were used as described previously [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEleven Em Ca cell lines (Ishikawa, Hec6, Hec50, Hec59, Hec88, Hec108, Hec116, Hec151, Hec155, Hec180, and Hec251), whcih we have established previously [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], were used. The full-length ALK expression plasmid or empty vector were transfected into Hec6 cells (which lack endogenous ALK expression) (Supplementary Figure S1A) and two stably overexpressing clones (H6-ALK#8 and H6-ALK#47) were established. ALK-knockdown lines (ALK-KD) were also generated using Hec59 cells (which have relatively high ALK expression) (Supplementary Figure S1B) and shRNA targeting the \u003cem\u003eALK\u003c/em\u003e gene (H59-shALK#11 and H59-shALK#33) as described previously [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTransfection\u003c/p\u003e \u003cp\u003eTransfection was carried out using LipofectAMINE PLUS (Invitrogen) as described previously [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eReverse transcription (RT)-PCR\u003c/p\u003e \u003cp\u003ecDNA was synthesized from 2 \u0026micro;g of total RNA. Amplification by RT-PCR was carried out in the exponential phase to allow comparisons between cDNA synthesized from identical reactions. Primers for the \u003cem\u003eALK\u003c/em\u003e and \u003cem\u003eGAPDH\u003c/em\u003e genes were used as described previously [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWestern blot assays\u003c/p\u003e \u003cp\u003eTotal cellular proteins were isolated using RIPA buffer [20 mM Tris-HCl (pH 7.2), 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate]. Aliquots of the proteins were resolved by SDS-PAGE, transferred to PVDF membranes, and probed with primary antibodies coupled to the ECL detection system (Amersham Pharmacia Biotechnology, Tokyo, Japan), as described previously [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]..\u003c/p\u003e \u003cp\u003eFlow cytometry and Aldefluor assay\u003c/p\u003e \u003cp\u003eCells were fixed using 70% alcohol and stained with propidium iodide (Sigma) for cell cycle analysis. ALDH1 enzyme activity in viable cells was determined using a fluorogenic dye-based Aldefluor assay (Stem Cell Technologies, Grenoble, France) according to the manufacturer\u0026rsquo;s instructions. The prepared cells were analyzed by flow cytometry using BD FACS Calibur (BD Biosciences) and CellQuest Pro software version 3.3 (BD Biosciences), as described previously [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]..\u003c/p\u003e \u003cp\u003eSpheroid assay\u003c/p\u003e \u003cp\u003eCells (x 10\u003csup\u003e3\u003c/sup\u003e) were plated in low cell binding plates (Thermo Fisher Scientific, Yokohama, Japan) in Cancer Stem Cell Premium (ProMab Biotech, Richmond, CA). Uniform spheroids of at least 50 \u0026micro;m in diameter were counted approximately two weeks after plating, as described previously [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]..\u003c/p\u003e \u003cp\u003eWound healing assay\u003c/p\u003e \u003cp\u003eCells were seeded into 24-well tissue culture plates, and grown to reach 90\u0026ndash;100% confluence. After a cell monolayer formed, a wound was scratched with a sterile 200-\u0026micro;l tip. The area of the wound was also analyzed using ImageJ software version 1.41. Closure of the wound was measured in pixels, and used as a measure of cell migration, as described previously [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]..\u003c/p\u003e \u003cp\u003eFIuorescence \u003cem\u003ein situ\u003c/em\u003e hybridization (FISH)\u003c/p\u003e \u003cp\u003eFor analysis of the \u003cem\u003eALK\u003c/em\u003e (2p23) locus, dual-color FISH studies were conducted on four HGSC cases with strong ALK immunopositivity using the Vysis LSI ALK break-apart rearrangement probe (Abbott Molecular, Abbott Park, IL, USA) according to the manufacturer\u0026rsquo;s instructions, as described previously [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]..\u003c/p\u003e \u003cp\u003eMutation analyses of the \u003cem\u003eALK\u003c/em\u003e gene\u003c/p\u003e \u003cp\u003eGenomic DNA was extracted from Hec59 cells using a Wizard Genomic DNA Purification kit (Promega, Madison, WT, USA) according to the manufacturer\u0026rsquo;s instructions. Mutation analyses of exons 20, 23, 24, and 25 of the \u003cem\u003eALK\u003c/em\u003e gene were carried out as described previously [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Briefly, the PCR products were purified using a NucleoSpin Gel and PCR Clean-up (Macherey-Nagel, D\u0026uuml;ren, Germany) and bidirectional sequencing was performed using the BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, USA) on the ABI3130 genetic analyzer (Applied Biosystems). Sequencing Analysis software v5.4 (Applied Biosystems) along with a manual chromatogram review was used for sequence analysis.\u003c/p\u003e \u003cp\u003eTCGA data analysis\u003c/p\u003e \u003cp\u003ecBioportal (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.cbioportal.org/\u003c/span\u003e\u003cspan address=\"http://www.cbioportal.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to extract The Cancer Genome Atlas (TCGA) HGSA ALK mutational data and expression data (RNA Seq V2 PSEM) associated with 506 Em Ca cases. ALK mRNA expression levels were subcategorized into \u0026lsquo;high\u0026rsquo; and \u0026lsquo;low\u0026rsquo; groups on the basis of the Z score (cutoff values were 0.5). Cancer Cell Line Encyclopedia (CCLE) data describing the \u003cem\u003eALK\u003c/em\u003e gene status and the relative expression of ALK mRNA in 28 Em Ca cell lines were also extracted from cBioPortal, as described previously [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]..\u003c/p\u003e \u003cp\u003eStatistics\u003c/p\u003e \u003cp\u003eComparative data were analyzed using the Mann-Whitney \u003cem\u003eU\u003c/em\u003e-test and Spearman\u0026rsquo;s correlation coefficient. Overall survival (OS) was calculated as the time between onset and death or the date of the last follow-up evaluation. Progression-free survival (PFS) was also examined from the onset of treatment until relapse, disease progression, or last follow-up evaluation. OS and PFS were estimated using the Kaplan-Meier method, and statistical comparisons were made using the log rank test. The cut-off for statistical significance was set as p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, as described previously [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]..\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eALK is overexpressed in a subset of Em Ca cases\u003c/p\u003e \u003cp\u003eRepresentative images of IHC findings for ALK in Em Ca are illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. Strong cytoplasmic ALK immunoreactivity was mainly observed in G3 Em Ca, in contrast to the weak membranous immunoreaction in G1 tumors. ALK immunopositivity was observed in 14 (15.4%) of 91 G1 Em Ca, 3 (7.5%) of 40 G2, and 17 (45.9%) of 37 G3; the difference between G3 scores and the other tumor grades was statistically significant (Supplementary Table S1). A similar finding was also observed with regard to average ALK scores (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), whereas there were no associations between ALK expression and other clinicopathological factors in Em Ca (Supplementary Table S1). ALK immunoreactivity clearly overlapped with ALK mRNA expression, and the ALK IHC scores were significantly higher in the ALK ISH-high category when compared to the ALK ISH-low group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). In addition, FISH did not reveal \u003cem\u003eALK\u003c/em\u003e rearrangement or amplification in four Em Ca cases with strong ALK immunoreactivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eAmong eleven Em Ca cell lines, two (Hec59 and Hec251) had relatively high ALK mRNA expression (Supplementary Figure S1A), in line with CCLE data analyses (Supplementary Figure S1B). However, ALK protein was only detected in Hec59 cells (Supplementary Figure S1C). Neither Hec59 nor Hec251 cells had mutations in exons 20, 23, 23, 24, and 25 of the \u003cem\u003eALK\u003c/em\u003e gene (Supplementary Figure S1D); these data are consistent with CCLE data that show there is no association between \u003cem\u003eALK\u003c/em\u003e gene mutation and levels of ALK mRNA (Supplementary Table S2).\u003c/p\u003e \u003cp\u003eTogether, these findings suggest that cytoplasmic ALK expression without chromosomal rearrangement, along with the increased mRNA expression, is observed together with increased ALK mRNA expression in G3 Em Ca.\u003c/p\u003e \u003cp\u003eALK overexpression increased cell proliferation and decreased susceptibility of apoptosis in Em Ca cells\u003c/p\u003e \u003cp\u003eTo determine whether ALK played a functional role in Em Ca cells, we first established two independent Hec6 cell line clones stably overexpressing full-length ALK and two independent Hec59 cell line clones in which ALK expression was blocked by an ALK-specific shRNA.\u003c/p\u003e \u003cp\u003eH6-ALK cells tended to proliferate rapidly than mock-transfected cells. This was consistent with the display of markers associated with cell cycle progression including increased G2/M fraction, increased Rb and pRb expression, and decreased p21\u003csup\u003ewaf1\u003c/sup\u003e expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In contrast, H59-shALK cells tended to proliferate more slowly, and exhibited a reduced S phase fraction, although the expression of several cell cycle-related markers remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eCompared with mock-transfected cells, ADR treatment of H6-ALK cells induced less cleaved caspase-3 expression and fewer apoptotic features, such as a less prominent sub-G1 FACS profile (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and Supplementary S2A,B). The opposite effects were observed in ADR-treated H59-shALK cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD and Supplementary Figure S2C,D).\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA shows representative IHC images for Ki-67 and cleaved PARP1 in ALK-high and ALK-low G3 Em Ca cases. There was a positive correlation between the two markers in Em Ca (Table\u0026nbsp;1). Both Ki-67 and cleaved PARP1 immunoreactivities were significantly higher in G3 Em Ca than in G1/G2 tumors. Average Ki-67 LI values were also significantly higher in ALK-positive tumors when compared with their ALK-negative counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), and ALK score was also positively and significantly correlated with Ki-67 LI (Table\u0026nbsp;1). However, there were no correlations between ALK levels and cleaved PARP1 score (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eThese findings suggest that ALK overexpression increases proliferation and reduces susceptibility to apoptosis in Em Ca cells.\u003c/p\u003e \u003cp\u003eALK overexpression enhances CSC features and migration in Em Ca cells\u003c/p\u003e \u003cp\u003eALK induces CSC features through activation of the epithelial-mesenchymal transition (EMT) in ovarian high-grade serous carcinomas and uterine carcinosarcoma [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. We therefore examined whether the association between ALK expression and CSC properties was also feature of Em Ca. Indeed, H6-ALK cells exhibited increased expression of several CSC and EMT markers including CD133, CD44s, Nestin, Sox2, Snail, Slug, and ZEB1 when compared to mock cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The Aldefluor assay also revealed a significant ALDH1\u003csup\u003ehigh\u003c/sup\u003e population in the H6-ALK cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), in line with a significant increase in the number of well-defined, round spheroids that were over 50 mm in diameter (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). In contrast, levels of these markers were significantly lower in H59-shALK cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD,E,F).\u003c/p\u003e \u003cp\u003eWe then examined whether ALK expression modulates cell motility using the wound healing assay. H6-ALK cells refilled wounded empty spaces more rapidly, and exhibited significantly increased migration capacity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Conversely, H59-shALK cells were less able to refill the empty wound spaces, and had lower migration rates than mock cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eSince ALK overexpression is associated with neuroendocrine (NE) differentiation and \u003cem\u003eTP53\u003c/em\u003e gene status in ovarian high-grade serous carcinomas and uterine carcinosarcoma [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], we investigated NE markers and p53 staining in Em Ca (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Distinct cytoplasmic and/or membranous immunostaining for CD56, Syn, CD133, and ALDH1, as well as nuclear immunoreactivity for p53, Sox2, and Sox7 were observed in Em Ca cells. Average IHC scores for CD56, Syn, p53, cytoplasmic (Cyt)-CD133, ALDH1, and Sox2, as well as ALK, were significantly higher in G3 Em Cas as compared to those of G1 or G2 tumors, but this differential pattern was not evident for membranous CD133 or Sox7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). In addition, ALK score was positively correlated with CD56, Syn, Cyt-CD133, ALDH1, Sox2, Sox7, and p53 scores. Both Sox2 and Cyt-CD133 scores were also positively associated with a subset of NE and CSC markers (Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eThese findings suggest that ALK overexpression enhances CSC properties, migration capability, and NE features in Em Ca cells.\u003c/p\u003e \u003cp\u003eCorrelation between ALK status and survival in Em Ca\u003c/p\u003e \u003cp\u003eKaplan-Meier curves showed that Em Ca patients with high ALK mRNA expression and wild-type \u003cem\u003eALK\u003c/em\u003e gene status had poorer OS and PFS when compared to patients with low ALK mRNA expression and mutant \u003cem\u003eALK\u003c/em\u003e (Supplementary Figure S3).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study clearly provides evidence of a positive correlation between ALK mRNA and protein in Em Ca tissues. Furthermore, we found no evidence of ALK chromosomal rearrangements or mutations in both Em Ca clinical samples or cell lines. We previously showed that full length ALK overexpression was under the transcriptional control of SoxB1 (Sox2 and Sox3) and SoxF (Sox7 and Sox17) in ovarian high-grade serous carcinoma [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], suggesting the existence of a positive feedback loop between ALK and Sox factors. We also found that \u003cem\u003eALK\u003c/em\u003e promoter activity was suppressed by wild-type p53, but not mutant p53 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In general, a high p53 score (60\u0026ndash;100% immunopositive cells) is sufficient to identify a p53 mutation in 94% of cases [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Given our present finding that ALK expression was positively correlated with Sox2, Sox7, and p53 scores in Em Ca tissues, we suggest that transcriptional upregulation of ALK may also occur in Em Ca via transcriptional activators including Sox factors and p53.\u003c/p\u003e \u003cp\u003eWe also found that ALK overexpression was significantly higher in G3 Em Ca when compared to G1/G2 tumors. ALK was predominantly cytoplasmic in G3 tumors, and membrane localized in G1/G2 tumors. Moreover, ALK expression was associated with increased proliferation but not with levels of cleaved PARP1 in Em Ca cells. This is consistent with evidence that cytoplasmic localization of the ALK tyrosine kinase domain promotes proliferation, whereas membrane attachment is critical for initiation of neurite outgrowth and proliferation arrest [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInterestingly, stable ALK overexpression in Em Ca cell lines significantly increased proliferation, reduced susceptibility to apoptosis, and these effects were abrogated in ALK-knockdown cells. Given that loss of ALK inhibits the growth of breast carcinoma cell lines [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], we suggest that cytoplasmic ALK modulates cell survival and proliferation. This is probably mediated via activation of ALK-related signaling pathways including phosphatidylinositol 3-kinase-AKT, Janus Kinase-STAT, and mitogen-activated protein kinase pathways, which contribute to regulation of cell cycle progression and apoptotic features [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAn important finding of this study is that ALK overexpression enhanced CSC properties; this is consistent with the upregulation of several CSC markers, including Cyt-CD133, ALDH1, and Sox2 in Em Ca tissues. Moreover, ALK overexpression significantly enhanced migration capability, whereas ALK knockdown abrogates this effect. In glioblastoma, CSCs are more invasive than non-CSCs [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Taking these results together, we infer that the increased migration capability of CSC-like cells may contribute to the aggressive features of ALK-overexpressing Em Ca cells.\u003c/p\u003e \u003cp\u003eFinally, overexpression of ALK, as well as Sox2, was also clearly associated with expression of NE markers in G3 Em Ca cells. Similar findings were also observed in ovarian high-grade serous carcinoma [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In general, the prognosis of NE carcinoma is poor, as more than 80% of patients present with metastatic disease at diagnosis, and there are few effective therapies for this cancer type [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Given that Sox2 blocks the differentiation of the neural progenitor population [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], we suggest that the combined activities of ALK and Sox2 induce an NE-like phenotype in Em Ca cells, and that this contributes to the poor OS and PFS in Em Ca. Our hypothesis was supported by analysis of TCGA data, which showed that high ALK mRNA expression and wild-type \u003cem\u003eALK\u003c/em\u003e status were associated with poorer prognosis in Em Ca cases.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur overall conceptual framework for the role of ALK in aggressive Em Ca is summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. Full-length ALK overexpression occurs in a subset of Em Ca, particularly in G3 tumors, and contributes to establishment and maintenance of aggressive phenotypic characteristics through modulation of several biological processes.\u003c/p\u003e "},{"header":"Abbreviations","content":" \u003cp\u003eALK, anaplastic lymphoma kinase; Em Ca, endometrial carcinoma; CSC, cancer stem cell; EMT, epithelial-mesenchymal transition; IHC, immunohistochemistry; NE, neuroendocrine\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAY, YN, and MS carried out the majority of the experiments, analyzed the data, and wrote the manuscript. They were helped by MH, YO, TM, MN, YI, TI, KO, YH, and NF. All authors reviewed and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by a grant from JSPS KAKENHI Grant Number 21K20781\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data sets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Kitasato University Medical Ethics Committee (B20-81) and was conducted according to the Helsinki Declaration. The requirement for informed consent was waived by the Kitasato University Hospital, as this was a retrospective study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declared no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eDepartment of Pathology, Kitasato University School of Medicine, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0374, Japan. \u003csup\u003e2\u003c/sup\u003eDepartment of Pathology, Kitasato University School of Allied Health Science, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0374, Japan.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRyan AJ, Susil B, Jobling TW, Oehler MK. Endometrial cancer. Cell Tissue Res. 2005;332:53\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKasoha M, Dernektsi C, Seibold A, Bohle RM, Takacs Z, Ioan-lulian I, Solomayer E-F, Juhasz-Boss I. Crosstalk of estrogen receptors and Wnt/β-catenin signaling in endometrial cancer. J Cancer Res Clin Oncol. 2020;146:315\u0026ndash;27.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUshijima K. Current status of gynecologic cancer in Japan. J Gynecol Oncol. 2009;20:67\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYamagami W, Mikami M, Nagase S, Tabata T, Kobayashi Y, Kaneuchi M, et al. Japan society of gynecologic oncology 2018 guidelines for treatment of uterine body neoplasms. J Gynecol Oncol. 2020;31:e18.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCragun JM, Havrilesky LJ, Calingaert B, Synan I, Secord AA, Soper JT, et al. Retrospective analysis of selective lymphoadenectomy in aapparent early-stage endometrial cancer. J Clin Oncol. 2005;23:3668\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eASTEC study group. Kitchener H, Swart AM, Qian Q, Amos C, Parmar MKB. Efficacy of systematic pelvic lymphoadenectomy in endometrial cancer (MRC ASTEC trial): a randomized study. Lancet. 2009;373:125\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBenedetti Panici P, Basile S, Maneschi F, Alberto Lissoni A, Signorelli M, Scambia G, et al. Systematic pelvic lymphoadenectomy vs. no lymphadenectomy in early-stage endometrial carcinoma: randomized clinical trial. J Natl Cancer Inst. 2008;100:1707\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSiegel RL, Miller KD, Jemal A. Cancer statistics. CA Cancer J Clin. 2016;66:7\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIwahara T, Fujimoto J, Wen D, Cupples R, Bucay N, Arakawa T, et al. Molecular characterization of ALK, a receptor tyrosine kinase expressed specifically in the nervous system. Oncogene. 1997;14:439\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorris SW, Naeve C, Mathew P, James PL, Kirstein MN, Cui X, et al. ALK, the chromosome 2 gene locus altered by the t(2;5) in non-Hodgkin\u0026rsquo;s lymphoma, encodes a novel neural receptor tyrosine kinase that is highly related to leukocyte tyrosine kinase (LTK). Oncogene. 1997;14:2175\u0026ndash;88.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBen-Neriah Y, Bauskin AR. Leukocytes express a novel gene encoding a putative transmembrane protein-kinase devoid of an extracellular domain. Nature. 1988;333:672\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaru Y, Hirai H, Takaku F. Human Itk: gene structure and preferential expression in human leukemic cells. Oncogene Res. 1990;5:199\u0026ndash;204.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBernards A, de la Monte SM. The Itk receptor tyrosine kinase is expressed in pre-B lymphocytes and cerebral neurons and uses a non-AUG translational initiator. EMBO J. 1990;2:2279\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheung NK, Dyer MA. Neuroblastoma: developmental biology, cancer genomics and immunotherapy. Nat Rev Cancer. 2013;13:397\u0026ndash;411.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReiff T, Huber L, Kramer M, Delattre O, Janoueix-Lerosey I, Rohrer H. Midkine and Alk signaling in sympathetic neuron proliferation and neuroblastoma predisposition. Development. 2011;13:685\u0026ndash;700.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHallberg B, Palmer RH. Mechanistic insight into ALK receptor tyrosine kinase in human cancer biology. Nat Rev Cancer. 2013;13:685\u0026ndash;700.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorris SW, Naeve C, Mathew P, James PL, Kirstein MN, Cui X, et al. ALK, the chromosome 2 gene locus altered by the t(2;5) in non-Hodgkin\u0026rsquo;s lymphoma, encodes a novel neural receptor tyrosine kinase that is highly related to leukocyte tyrosine kinase (LTK). Oncogene. 1997;14:2175\u0026ndash;88.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakeuchi K, Soda M, Togashi Y, Suzuki R, Sakata S, Hatano S, et al. RET, ROS1 and ALK fusions in lung cancer. Nat Med. 2012;18:378\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatsumoto T, Oda Y, Hasegawa Y, Hashimura M, Oguri Y, Inoue H, et al. Anaplastic lymphoma Kinase overexpression is associated with aggressive phenotypic characteristics of ovarian high-grade serous carcinoma. Am J Pathol. 191:1837\u0026ndash;1850.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eInoue H, Hashimura M, Akiya M, Chiba R, Saegusa M. Functional role of ALK-related signal cascades on modulation of epithelial-mesenchymal transition and apoptosis in uterine carcinosarcoma. Mol Cancer. 2017;16:37.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZaino R, Carinelli SG, Ellenson LH, Eng C, Katabuchi H, Konishi I, et al Tumours of the uterine corpus. In: Kurman RJ, Carcangiu ML, Herrington CS, Young RH, editors. WHO classification of tumours of female reproductive organs. Lyon: IARC; 2014. P.121 \u0026ndash; 54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCreasman W. Revised FIGO staging for carcinoma of the endometrium. Int J Gynaecol Obstet. 2009;105:109.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKuramoto H, Hamano M, Imai M, Fijisawa T, Kamata Y, Arai T, et al. Hec-1 cells: establishment of an \u003cem\u003ein vitro\u003c/em\u003e experimental system in endometrial carcinoma. Cell and molecular biology of endometrial carcinoma. Edited by Kuramoto H, Nishida M. Tokyo, Springer-Verlag, 2003, p.\u0026nbsp;3\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNishida M. Ishikawa cells: opening of in vitro hormone research on endometrial carcinoma. Cell and molecular biology of endometrial carcinoma. Edited by Kuramoto H, Nishida M. Tokyo, Springer-Verlag, 2003, p.\u0026nbsp;35\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChiba R, Akiya M, Hashimura M, Oguri Y, Inukai M, Hara A, et al. ALK signaling cascade confers multiple advantages to glioblastoma cells through neovascularization and cell proliferation. PLoS ONE. 2017;12:e0183526.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYemelyanova A, Vang R, Kshirsagar M, Lu D, Marks MA, Shih leM, Kurman RJ. Immunohistochemical staining patterns of p53 can serve as a surrogate marker for \u003cem\u003eTP53\u003c/em\u003e mutations in ovarian carcinomas: an immunohistochemical and nucleotide sequencing analysis. Mod Pathol. 2011;24:1248\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGouzi JY, Moog-Lutz C, Vigny M, Brunet-de Carvalho N. Role of the subcellular localization of ALK tyrosine kinase domain in neuronal differentiation of PC12 cells. J Cell Sci. 2005;118:5811\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSiraj AK, Beg S, Jehan Z, Prabhakaran S, Ahmed M, R.Hussain A, et al. ALK alteration is a frequent event in aggressive breast cancers. Breast Cancer Res. 2015;17:127.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHallberg B, Palmer RH. The role of the ALK receptor in cancer biology. Ann Oncol. 2016;27:iii4\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVolovetz J, Berezovsky AD, Alban T, Chen Y, Lauko A, Aranjuez GF, et al. Identifying conserved molecular targets required for cell migration of glioblastoma cancer stem cells. Cell Death Dis. 2020;11:152.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFreis P, Graillot E, Rousset P, Hervieu V, Chardon L, Lombard-Bohas C, et al. Prognostic factors in neuroendocrine carcinoma: biological markers are more useful than histomorphological markers. Sci Rep. 2017;7:40609.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKiefer JC. Back to basics. \u003cem\u003eSox\u003c/em\u003e genes. Dev Dyn. 2007;236:2356\u0026ndash;66.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"ALK, cancer stem cell, epithelial-mesenchymal transition, Neuroendocrine differentiation, endometrial carcinoma","lastPublishedDoi":"10.21203/rs.3.rs-2203804/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2203804/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eAlthough anaplastic lymphoma kinase (ALK) overexpression is overexpressed in several primary solid tumor types, its role in endometrial carcinoma (Em Ca) remains unclear.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe evaluated expression of ALK and its related molecules in clinical samples consisting of 168 Em Ca tissues. We also used Em Ca cell lines to evaluate the functional role of ALK.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eCytoplasmic ALK immunoreactivity in the absence of chromosomal rearrangement was positively correlated with ALK mRNA expression, and was significantly higher in Grade (G) 3 Em Ca than in G1 or G2 tumors. ALK immunoreactivity was also significantly associated with expression of cancer stem cell (CSC)-related molecules (cytoplasmic CD133, ALDH1, Sox2) and neuroendocrine markers (CD56 and synaptophysin). Although the proliferative index was significantly higher in ALK-positive Em Ca when compared to ALK- negative malignancies, there was no association between ALK expression and other clinicopathological factors in this disease. In Em Ca cell lines, full-length ALK overexpression increased proliferation, decreased susceptibility to apoptosis, enhanced cancer stem cell features, and accelerated cell mobility, whereas these phenotypes were abrogated in ALK-knockdown cells. Finally, tumors with either wild-type \u003cem\u003eALK\u003c/em\u003e or high ALK mRNA expression were associated with a poorer prognosis when compared to Em Ca with either mutant \u003cem\u003eALK\u003c/em\u003e or low ALK mRNA expression.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eFull-length ALK overexpression occurs in a subset of Em Ca, particularly in G3 tumors, and contributes to establishment and maintenance of aggressive phenotypic characteristics through modulation of several biological processes.\u003c/p\u003e","manuscriptTitle":"Anaplastic lymphoma kinase overexpression enhances an aggressive phenotypic characteristics of endometrial carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-12-05 16:06:30","doi":"10.21203/rs.3.rs-2203804/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2023-04-25T04:19:24+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-03-11T05:40:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"63c255dc-20c1-45d5-9a7e-c92be2808314","date":"2023-03-03T15:11:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"995ed0aa-c4c6-4326-9c1a-b6c544d9f043","date":"2023-03-03T15:07:05+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-02-27T22:42:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-02-17T14:38:42+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2022-12-01T23:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-12-01T22:57:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Cancer","date":"2022-10-25T21:34:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3d46f7fa-2048-4684-896c-a27bfca8367a","owner":[],"postedDate":"December 5th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-16T22:14:36+00:00","versionOfRecord":{"articleIdentity":"rs-2203804","link":"https://doi.org/10.1186/s12885-023-11144-2","journal":{"identity":"bmc-cancer","isVorOnly":false,"title":"BMC Cancer"},"publishedOn":"2023-08-17 22:02:52","publishedOnDateReadable":"August 17th, 2023"},"versionCreatedAt":"2022-12-05 16:06:30","video":"","vorDoi":"10.1186/s12885-023-11144-2","vorDoiUrl":"https://doi.org/10.1186/s12885-023-11144-2","workflowStages":[]},"version":"v1","identity":"rs-2203804","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2203804","identity":"rs-2203804","version":["v1"]},"buildId":"ehx78VzkSd0WSzXnipQa-","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}