Restricted accessResearch articleFirst published online 2021-07
Pro64His (rs4644) Polymorphism Within Galectin-3 Is a Risk Factor of Differentiated Thyroid Carcinoma and Affects the Transcriptome of Thyrocytes Engineered via CRISPR/Cas9 System
Galectin-3 (LGALS3) is an important glycoprotein involved in the malignant transformation of thyrocytes acting in the extracellular matrix, cytoplasm, and nucleus where it regulates TTF-1 and TCF4 transcription factors. Within LGALS3 gene, a common single-nucleotide polymorphism (SNP) (c.191C>A, p.Pro64His; rs4644) encoding for the variant Proline to Histidine at codon 64 has been extensively studied. However, data on rs4644 in the context of thyroid cancer are lacking. Thus, the aim of the present work was to evaluate the role of the rs4644 SNP as risk factor for differentiated thyroid cancer (DTC) and to determine the effect on the transcriptome in thyrocytes.
Methods:
A case/control association study in 1223 controls and 1142 unrelated consecutive DTC patients was carried out to evaluate the association between rs4644-P64H and the risk of DTC. We used the nonmalignant cell line Nthy-Ori (rs4644-C/A) and the CRISPR/Cas9 technique to generate isogenic cells carrying either the rs4644-A/A or rs4644-C/C homozygosis. Then, the transcriptome of the derivative and unmodified parental cells was analyzed by RNA-seq. Genes differentially expressed were validated by quantitative reverse transcription PCR and further tested in the parental Nthy-Ori cells after LGALS3 gene silencing, to investigate whether the expression of target genes was dependent on galectin-3 levels.
Results:
rs4644 AA genotype was associated with a reduced risk of DTC (compared with CC, ORadj = 0.66; 95% confidence interval = 0.46–0.93; Pass = 0.02). We found that rs4644 affects galectin-3 as a transcriptional coregulator. Among 34 genes affected by rs4644, HES1, HSPA6, SPC24, and NHS were of particular interest since their expression was rs4644-dependent (CC>AA for the first and AA>CC for the others), also in 574 thyroid tissues of Genotype-Tissue Expression (GTEx) biobank. Moreover, the expression of these genes was regulated by LGALS3-silencing. Using the proximity ligation assay in Nthy-Ori cells, we found that the TTF-1 interaction was genotype dependent.
Conclusions:
Our data show that in thyroid, rs4644 is a trans-expression quantitative trait locus that can modify the transcriptional expression of downstream genes, through the modulation of TTF-1.
Get full access to this article
View all access options for this article.
References
1.
OlsonE, WintheiserG, WolfeKM, DroesslerJ, SilbersteinPT. 2019. Epidemiology of thyroid cancer: a review of the National Cancer Database, 2000–2013. Cureus, 11:e4127.
2.
GudmundssonJ, SulemP, GudbjartssonDF, JonassonJG, SigurdssonJ, BergthorssonJT, HeH, BlondalT, GellerF, JakobsdottirM, MagnusdottirDN, MatthiasdottirS, StaceySN, SkarphedinssonOB, HelgadottirH, LiW, NagyR, AguilloE, FaureE, PratsE, SaezB, MartinezM, EyjolfssonGI, BjornsdottirUS, HolmH, KristjanssonK, FriggeML, KristvinssonH, GulcherJR, JonssonT, RafnarT, HjartarssonH, MayordomoJI, de la ChapelleA, HrafnkelssonJ, ThorsteinsdottirU, KongA, StefanssonK. 2009. Common variants on 9q22.33 and 14q13.3 predispose to thyroid cancer in European populations. Nat Genet, 41:460–464.
WeiWJ, LuZW, WangY, ZhuYX, WangYL, JiQH. 2015. Clinical significance of papillary thyroid cancer risk loci identified by genome-wide association studies. Cancer Genet, 208:68–75.
5.
ŚwierniakM, WójcickaA, CzetwertyńskaM, DługosińskaJ, StachlewskaE, GierlikowskiW, KotA, GórnickaB, KoperskiL, BogdańskaM, WiechnoW, JażdżewskiK. 2016. Association between GWAS-derived rs966423 genetic variant and overall mortality in patients with differentiated thyroid cancer. Clin Cancer Res, 22:1111–1119.
6.
FiglioliG, EliseiR, RomeiC, MelaiuO, CipolliniM, BambiF, ChenB, KöhlerA, CristaudoA, HemminkiK, GemignaniF, FörstiA, LandiS. 2016. A comprehensive meta-analysis of case-control association studies to evaluate polymorphisms associated with the risk of differentiated thyroid carcinoma. Cancer Epidemiol Biomarkers Prev, 25:700–713.
7.
ZukO, HechterE, SunyaevSR, LanderES. 2012. The mystery of missing heritability: genetic interactions create phantom heritability. Acad Proc Natl Sci U S A, 109:1193–1198.
8.
SciacchitanoS, LavraL, MorganteA, UlivieriA, MagiF, De FrancescoGP, BellottiC, SalehiLB, Ricci A 2018 Galectin-3: one molecule for an alphabet of diseases, from A to Z. Int J Mol Sci 19:pii: E379.
9.
SaussezS, GlinoerD, ChantrainG, PattouF, CarnailleB, AndrèS, GabiusH-J, LaurentG. 2008. Serum galectin-1 and galectin-3 levels in benign and malignant nodular thyroid disease. Thyroid, 18:705–712.
10.
LiJ, VasilyevaE, WisemanSM. 2019. Beyond immunohistochemistry and immunocytochemistry: a current perspective on galectin-3 and thyroid cancer. Expert Rev Anticancer Ther, 19:1017–1027.
11.
CastronovoV, Van Den BrûleFA, JackersP, ClausseN, LiuFT, GilletC, SobelME. 1996. Decreased expression of galectin-3 is associated with progression of human breast cancer. J Pathol, 179:43–48.
12.
van den BruleFA, BuicuC, BerchuckA, BastRC, DeprezM, LiuFT, CooperDN, PietersC, SobelME, CastronovoV. 1996. Expression of the 67-kD laminin receptor, galectin-1, and galectin-3 in advanced human uterine adenocarcinoma. Hum Pathol, 27:1185–1191.
13.
IrimuraT, MatsushitaY, SuttonRC, CarraleroD, OhannesianDW, ClearyKR, OtaDM, NicolsonGL, LotanR. 1991. Increased content of an endogenous lactose-binding lectin in human colorectal carcinoma progressed to metastatic stages. Cancer Res, 51:387–393.
14.
SchoeppnerHL, RazA, HoSB, BresalierRS. 1995. Expression of an endogenous galactose-binding lectin correlates with neoplastic progression in the colon. Cancer, 75:2818–2826.
15.
SongS, JiB, RamachandranV, WangH, HafleyM, LogsdonC, BresalierRS. 2012. Overexpressed galectin-3 in pancreatic cancer induces cell proliferation and invasion by binding Ras and activating Ras signaling. PLoS One, 7:e42699.
16.
LotanR, BelloniPN, TresslerRJ, LotanD, XuXC, NicolsonGL. 1994. Expression of galectins on microvessel endothelial cells and their involvement in tumour cell adhesion. Glycoconj J, 11:462–468.
InoharaH, RazA. 1994. Identification of human melanoma cellular and secreted ligands for galectin-3. Biochem Biophys Res Commun, 201:1366–1375.
19.
XuXC, el-NaggarAK, LotanR. 1995. Differential expression of galectin-1 and galectin-3 in thyroid tumors. Potential diagnostic implications. Am J Pathol, 147:815–822.
ItoY, YoshidaH, TomodaC, MiyaA, KobayashiK, MatsuzukaF, YasuokaH, KakudoK, InoharaH, KumaK, MiyauchiA. 2005. Galectin-3 expression in follicular tumours: an immunohistochemical study of its use as a marker of follicular carcinoma. Pathology, 37:296–298.
22.
Elad-SfadiaG, HaklaiR, BalanE, KloogY. 2004. Galectin-3 augments K-Ras activation and triggers a Ras signal that attenuates ERK but not phosphoinositide 3-kinase activity. J Biol Chem, 279:34922–34930.
23.
LevyR, Grafi-CohenM, KraiemZ, KloogY. 2010. Galectin-3 promotes chronic activation of K-Ras and differentiation block in malignant thyroid carcinomas. Mol Cancer Ther, 9:2208–2219.
24.
Shalom-FeuersteinR, PlowmanSJ, RotblatB, AriottiN, TianT, HancockJF, KloogY. 2008. K-ras nanoclustering is subverted by overexpression of the scaffold protein galectin-3. Cancer Res, 68:6608–6616.
ParonI, ScaloniA, PinesA, BachiA, LiuFT, PuppinC, PandolfiM, LeddaL, Di LoretoC, DamanteG, TellG. 2003. Nuclear localization of Galectin-3 in transformed thyroid cells: a role in transcriptional regulation. Biochem Biophys Res Commun, 302:545–553.
27.
DagherSF, WangJL, PattersonRJ. 1995. Identification of galectin-3 as a factor in pre-mRNA splicing. Proc Natl Acad Sci U S A, 92:1213–1217.
28.
HaudekKC, SpronkKJ, VossPG, PattersonRJ, WangJL, ArnoysEJ. 2010. Dynamics of galectin-3 in the nucleus and cytoplasm. Biochim Biophys Acta, 1800:181–189.
29.
FritschK, MernbergerM, NistA, StieweT, BrehmA, JacobR. 2016. Galectin-3 interacts with components of the nuclear ribonucleoprotein complex. BMC Cancer, 16:502.
30.
NakaharaS, RazA. 2007. Regulation of cancer-related gene expression by galectin-3 and the molecular mechanism of its nuclear import pathway. Cancer Metastasis Rev, 26:605–610.
31.
SongS, ByrdJC, MazurekN, LiuK, KooJS, BresalierRS. 2005. Galectin-3 modulates MUC2 mucin expression in human colon cancer cells at the level of transcription via AP-1 activation. Gastroenterology, 129:1581–1591.
32.
LinHM, PestellRG, RazA, KimHR. 2002. Galectin-3 enhances cyclin D(1) promoter activity through SP1 and a cAMP-responsive element in human breast epithelial cells. Oncogene, 21:8001–8010.
33.
LiuFT, RabinovichGA. 2005. Galectins as modulators of tumour progression. Nat Rev Cancer, 5:29–41.
34.
ShimuraT, TakenakaY, TsutsumiS, HoganV, KikuchiA, RazA. 2004. Galectin-3, a novel binding partner of beta-catenin. Cancer Res, 64:6363–6367.
35.
LeeYK, LinTH, ChangCF, LoYL. 2013. Galectin-3 silencing inhibits epirubicin-induced ATP binding cassette transporters and activates the mitochondrial apoptosis pathway via β-catenin/GSK-3β modulation in colorectal carcinoma. PLoS One, 8:e82478.
36.
KimHR, LinHM, BiliranH, RazA. 1999. Cell cycle arrest and inhibition of anoikis by galectin-3 in human breast epithelial cells. Cancer Res, 59:4148–4154.
37.
WeinbergerPM, AdamBL, GourinCG, MoretzWH3rd, BollagRJ, WangBY, LiuZ, LeeJR, TerrisDJ. 2007. Association of nuclear, cytoplasmic expression of galectin-3 with beta-catenin/Wnt-pathway activation in thyroid carcinoma. Arch Otolaryngol Head Neck Surg, 133:503–510.
38.
ShimuraT, TakenakaY, FukumoriT, TsutsumiS, OkadaK, HoganV, KikuchiA, KuwanoH, RazA. 2005. Implication of galectin-3 in Wnt signaling. Cancer Res, 65:3535–3537.
39.
Gilbert-SirieixM, MakoukjiJ, KimuraS, TalbotM, CaillouB, MassaadC, Massaad-MassadeL. 2011. Wnt/β-catenin signaling pathway is a direct enhancer of thyroid transcription factor-1 in human papillary thyroid carcinoma cells. PLoS One, 6:e22280.
40.
GuazziS, PriceM, De FeliceM, DamanteG, MatteiMG, Di LauroR. 1990. Thyroid nuclear factor 1 (TTF-1) contains a homeodomain and displays a novel DNA binding specificity. EMBO J, 9:3631–3639.
41.
TakenakaY, InoharaH, YoshiiT, OshimaK, NakaharaS, AkahaniS, HonjoY, YamamotoY, RazA, KuboT. 2003. Malignant transformation of thyroid follicular cells by galectin-3. Cancer Lett, 195:111–119.
42.
Nangia-MakkerP, RazT, TaitL, HoganV, FridmanR, RazA. 2007. Galectin-3 cleavage: a novel surrogate marker for matrix metalloproteinase activity in growing breast cancers. Cancer Res, 67:11760–11768.
43.
Nangia-MakkerP, WangY, RazT, TaitL, BalanV, HoganV, RazA. 2010. Cleavage of galectin-3 by matrix metalloproteases induces angiogenesis in breast cancer. Int J Cancer, 127:2530–2541.
44.
MazurekN, ByrdJC, SunY, UenoS, BresalierRS. 2011. A galectin-3 sequence polymorphism confers TRAIL sensitivity to human breast cancer cells. Cancer, 117:4375–4380.
45.
Nangia-MakkerP, HonjoY, SarvisR, AkahaniS, HoganV, PientaKJ, RazA. 2000. Galectin-3 induces endothelial cell morphogenesis and angiogenesis. Am J Pathol, 156:899–909.
46.
BalanV, Nangia-MakkerP, SchwartzAG, JungYS, TaitL, HoganV, RazT, WangY, YangZQ, WuGS, GuoY, LiH, AbramsJ, CouchFJ, LingleWL, LloydRV, EthierSP, TainskyMA, RazA. 2008. Racial disparity in breast cancer and functional germ line mutation in galectin-3 (rs4644): a pilot study. Cancer Res, 68:10045–10050.
47.
FangSQ, FengYM, LiM. 2017. Correlations of galectin-3 gene polymorphisms with risk and prognosis of cervical cancer in Chinese populations: a case-control study. Oncol Res Treat, 40:533–539.
48.
MeyerA, CoinacI, BogdanovaN, DubrowinskajaN, TurmanovN, HauboldS, SchürmannP, ImkampF, von KlotC, MerseburgerAS, MachtensS, BremerM, HillemannsP, KuczykMA, KarstensJH, SerthJ, DörkT. 2013. Apoptosis gene polymorphisms and risk of prostate cancer: a hospital-based study of German patients treated with brachytherapy. Urol Oncol, 31:74–81.
49.
CiregiaF, BuglianiM, RonciM, GiustiL, BoldriniC, MazzoniMR, MossutoS, GranoF, CnopM, MarselliL, GiannacciniG, UrbaniA, LucacchiniA, MarchettiP. 2017. Palmitate-induced lipotoxicity alters acetylation of multiple proteins in clonal β cells and human pancreatic islets. Sci Rep, 7:13445.
50.
KimSJ, ShinJY, CheongTC, ChoiIJ, LeeYS, ParkSH, ChunKH. 2011. Galectin-3 germline variant at position 191 enhances nuclear accumulation and activation of β-catenin in gastric cancer. Clin Exp Metastasis, 28:743–750.
51.
LeeYH, SongGG. 2015. Genome-wide pathway analysis in glioma. Neoplasma, 62:230–238.
52.
NishikawaY, KodamaY, ShiokawaM, MatsumoriT, MaruiS, KuriyamaK, KuwadaT, SogabeY, KakiuchiN, TomonoT, MimaA, MoritaT, UedaT, TsudaM, YamauchiY, SakumaY, OtaY, MarunoT, UzaN, UesugiM, KageyamaR, ChibaT, SenoH. 2019. Hes1 plays an essential role in Kras-driven pancreatic tumorigenesis. Oncogene, 38:4283–4296.
53.
LiuA, ZhangD, YangX, SongY. 2019. Estrogen receptor alpha activates MAPK signaling pathway to promote the development of endometrial cancer. J Cell Biochem, 120:17593–17601.
54.
LiTT, LiuMR, PeiDS. 2019. Friend or foe, the role of EGR-1 in cancer. Med Oncol, 37:7.
55.
LiL, AmeriAH, WangS, JanssonKH, CaseyOM, YangQ, BeshiriML, FangL, LakeRG, AgarwalS, AlilinAN, XuW, YinJJ, KellyK. 2019. EGR1 regulates angiogenic and osteoclastogenic factors in prostate cancer and promotes metastasis. Oncogene, 38:6241–6255.
56.
Degl'InnocentiD, RomeoP, TarantinoE, SensiM, CassinelliG, CatalanoV, LanziC, PerroneF, PilottiS, SeregniE, PierottiMA, GrecoA, BorrelloMG. 2013. DUSP6/MKP3 is overexpressed in papillary and poorly differentiated thyroid carcinoma and contributes to neoplastic properties of thyroid cancer cells. Endocr Relat Cancer, 20:23–37.
57.
AhmadMK, AbdollahNA, ShafieNH, YusofNM, RazakSRA. 2018. Dual-specificity phosphatase 6 (DUSP6): a review of its molecular characteristics and clinical relevance in cancer. Cancer Biol Med, 15:14–28.
58.
LongqiuY, PengchengL, XuejieF, PengZ. 2016. A miRNAs panel promotes the proliferation and invasion of colorectal cancer cells by targeting GABBR1. Cancer Med, 5:2022–2031.
59.
LouI, OdoricoS, YuXM, HarrisonA, Jaskula-SztulR, ChenH. 2018. Notch3 as a novel therapeutic target in metastatic medullary thyroid cancer. Surgery, 163:104–111.
60.
HuangZ, ChengY, ChiuPM, CheungFMF, NichollsJM, KwongDL-W, LeeAWM, ZabarovskyER, StanbridgeEJ, LungHL, LungML. 2012. Tumor suppressor Alpha B-crystallin (CRYAB) associates with the cadherin/catenin adherens junction and impairs NPC progression-associated properties. Oncogene, 31:3709–3720.
61.
RuanH, LiY, WangX, SunB, FangW, JiangS, LiangC. 2020. CRYAB inhibits migration and invasion of bladder cancer cells through the PI3K/AKT and ERK pathways. Jpn J Clin Oncol, 50:254–260.
62.
SunX, ZouT, ZuoC, ZhangM, _ShiB, JiangZ, CuiH, LiaoX, LiX, TangY, LiuY, Liu × 2018. IL-1α inhibits proliferation and adipogenic differentiation of human adipose-derived mesenchymal stem cells through NF-κB- and ERK1/2-mediated proinflammatory cytokines. Cell Biol Int, 42:794–803.
63.
SigurdsonAJ, BrennerAV, RoachJA, GoudevaL, MüllerJA, NerlichK, ReinersC, SchwabR, PfeifferL, WaldenbergerM, BraganzaM, XuL, SturgisEM, YeagerM, ChanockSJ, PfeifferRM, AbendM, PortM. 2016. Selected single-nucleotide polymorphisms in FOXE1, SERPINA5, FTO, EVPL, TICAM1 and SCARB1 are associated with papillary and follicular thyroid cancer risk: replication study in a German population. Carcinogenesis, 37:677–684.
64.
YinH, MengT, ZhouL, ChenH, SongD. 2017. SPC24 is critical for anaplastic thyroid cancer progression. Oncotarget, 8:21884–21891.
65.
GaoF, HuangW, ZhangY, TangS, ZhengL, MaF, WangY, TangH, Li × 2015. Hes1 promotes cell proliferation and migration by activating Bmi-1 and PTEN/Akt/GSK3β pathway in human colon cancer. Oncotarget, 6:38667–38680.
66.
LiX, CaoY, LiM, JinF. 2018. Upregulation of HES1 promotes cell proliferation and invasion in breast cancer as a prognosis marker and therapy target via the AKT pathway and EMT process. J Cancer, 9:757–766.
67.
YamashitaAS, GeraldoMV, FuziwaraCS, KulcsarMA, FrigugliettiCU, da CostaRB, BaiaGS, KimuraET. 2013. Notch pathway is activated by MAPK signaling and influences papillary thyroid cancer proliferation. Transl Oncol, 6:197–205.
68.
LeeJ, SeolMY, JeongS, KwonHJ, LeeCR, KuCR, KangSW, JeongJJ, ShinDY, NamKH, LeeEJ, ChungWY, JoYS. 2015. KSR1 is coordinately regulated with Notch signaling and oxidative phosphorylation in thyroid cancer. J Mol Endocrinol, 54:115–124.
69.
SomnayYR, YuXM, LloydRV, LeversonG, AburjaniaZ, JangS, Jaskula-SztulR, ChenH. 2017. Notch3 expression correlates with thyroid cancer differentiation, induces apoptosis, and predicts disease prognosis. Cancer, 123:769–782.
