Page 160 - 2022_01-Haematologica-web

S.J. Erkeland et al.
ecules that are active in the TGFb pathway or due to dis- ruption of the network by oncoproteins.43 We determined miR-200c and miR-141-mediated downregulation of TGFbR3 transcripts in HeLa cells. TGFbR3 is a context- dependent regulator of TGFb signaling and a potent tumor suppressor that is frequently inactivated in various types of cancer.32,34,44 In agreement with the gene expression data, we observed reduced TGFbR3 membrane expres- sion on T-PLL cells. Although the levels of TGFbR1 mRNA and protein were unaffected, we did observe reduced TGFbR1 membrane expression on T-PLL cells. Reduced TGFbR3 expression in HeLa did not affect SMAD2/3 lev- els, though we noted an aberrant p-SMAD2 and p- SMAD3 level. Furthermore, we found that T-PLL samples with high miR-200c/141 levels have decreased p-SMAD2 and p-SMAD3 levels. Aberrant p-SMAD3 plays critical roles in stemness of tumor cells and progression of malig- nancies.45,46 Although this would suggest an important role of p-SMAD proteins in T-PLL leukemogenesis, the oncogenic downstream functions of aberrant TGFbR3 expression and p-SMAD in T-PLL remain to be further investigated.
ZEB1 and ZEB2 are highly related SMAD-interacting transcription factors and important players in the induc- tion of the epithelial–mesenchymal transition, which pro- motes invasion of tumor cells into the surrounding tissues, drug resistance, cell survival and metastasis.47 ZEB2 binds to SMAD2/3 and to SMAD1/5/8 in ligand-stimulated cells.31,48 ZEB proteins are involved in transcriptional repression by interacting with co-repressor protein CtBP and the NuRD chromatin-remodeling corepressor com- plex and are transcriptional activators by binding to P300/PCAF.49 ZEB1 and ZEB2 contain multiple miR-200c/141 target sequences in their 3’-UTR.30 Endogenous miRNA levels strongly repress ZEB1 and/or ZEB2 expression in a cell type-dependent fashion. For instance, during EMT miR-200 family members repress translation of both ZEB1 and ZEB2, whereas in CD8+ T cells only ZEB2 levels are downregulated, a process that is presumably controlled by RNA-binding proteins (RBP).30 Overexpression of miR-200 in CD8+ T cells selectively repressed ZEB2 expression, but not ZEB1, and promoted memory T-cell development.30 Interestingly, we found strong ZEB2 downregulation in T-PLL, whereas ZEB1 expression was not affected. The mechanism behind the selective downregulation of ZEB2 in T-PLL cells most-like- ly involves RBP.30 However, the RBP involved in selective ZEB expression regulation remain to be identified. How downregulation of ZEB2 and the above discussed aberrant p-SMAD3 levels affect target gene expression in T-PLL and how this contributes to oncogenic transformation remains to be unraveled.
We also found a strong upregulation of miR-181a and miR-181b in a subset of T-PLL samples. Upregulation of miR-181 is reported in human cancer, e.g., breast cancer and chronic lymphocytic leukemia (CLL).50-52 Although miR-181 is frequently downregulated in CLL as a whole, enhanced expression of miR-181a is associated with dis- ease progression in trisomy 12 CLL cases.52 However, as this chromosomal aberration is not found in T-PLL,2 miR- 181 is presumably upregulated by a different mechanism. For instance, there is strong evidence that stress-induced
STAT3 enhances miR-181 expression in tumor cells,53 a signaling cascade that is commonly activated in T-PLL.2 Enhanced miR-181 expression may have oncogenic trans- forming functions in T-PLL by affecting multiple path- ways. Ectopic expression of miR-181a in normal mouse CD4+ T cells augments the strength and sensitivity of TCR signaling to agonists.54 In normal T-cell development miR-181 controls expression of various phosphatases, such as SHP2, PTPN22, DUSP5, DUSP6 and NRARP, thereby enhancing NOTCH and TCR signaling.29 In agreement, we found a significant downregulation of DUSP5 mRNA expression in T-PLL cells, which may be caused by miR-181-mediated repression. The importance of miR-181 for T-cell leukemogenesis is further demon- strated by an experiment in which genomic deletion of MIR181A1/B1 specifically inhibits NOTCH-induced leukemogenesis in mice through repression of down- stream feedback mechanisms of NOTCH and pre-TCR signaling.29 Thus, overexpression of miR-181a1/b1 may contribute to enhanced NOTCH and TCR signaling, thereby enhancing oncogenesis in T-PLL.
In conclusion, we have shown that based on miRNA expression T-PLL is most similar to effector T-cell subsets albeit with some degree of heterogeneity between T-PLL cases. A set of 35 miRNA is aberrantly expressed in T-PLL, with miR-200c/141 being the most upregulated cluster. Aberrant expression of miR-200c/141 affects TGFb-con- trolled mechanisms that may contribute to the pathogen- esis of T-PLL. Additionally, the extent of overexpression of miR-200c and miR-141 appeared to significantly correlate with increased white blood cell counts and poor survival and may thus have prognostic value. Our data highlight a potential role for aberrantly expressed oncogenic miRNA in the pathogenesis of T-PLL, which may pave the way for new therapeutic approaches in this disease.
Disclosures
No conflicts of interest to disclose.
Contributions
SJE and AWL are corresponding authors; SJE, AWL, KL and VHJV designed the study; SJE, AWL and CJS wrote the manu- script; MCKK collected clinical data; KL performed cytogenetics and morphology analyses; JSV performed gene expression profil- ing, T-cell receptor gene rearrangement analysis, immuno-pheno- typing and FACsorting; GDC performed western blotting; CJS and YMM performed flowcytometry and miRNA experiments; IVZ, CJS, AHB, performed molecular cloning, cDNA library preparations and miRNA expression experiments; EMB per- formed next-generation sequencing; HVW, LGL and DR per- formed bioinformatic analyses.
Acknowledgments
We thank Steven Koetzier, Fabiënne van Opstal, Bernard Stikker and Stijn van den Broek for technical assistance during their bachelor and master projects.
Funding
This work was supported an unrestricted grant from Bayer, Mijdrecht, the Netherlands (to MCKK, KvL, and AWL). CJS and IVZ are supported by Dutch Cancer Society (KWF), grant number: 10948.
152
haematologica | 2022; 107(1)