HMGA1 negatively regulates NUMB expression at transcriptional and post transcriptional level in glioblastoma stem cells.

Glioblastoma (GBM) is a lethal, fast-growing brain cancer, affecting 2-3 per 100,000 adults per year. It arises from multipotent neural stem cells which have reduced their ability to divide asymmetrically and hence divide symmetrically, generating increasing number of cancer stem cells, fostering tumor growth. We have previously demonstrated that the architectural transcription factor HMGA1 is highly expressed in brain tumor stem cells (BTSCs) and that its silencing increases stem cell quiescence, reduces self-renewal and sphere-forming efficiency in serial passages, suggesting a shift from symmetric to asymmetric division. Since NUMB expression is fundamental for the fulfillment of asymmetric division in stem cells, and is lost or reduced in many tumors, including GBM, we have investigated the ability of HMGA1 to regulate NUMB expression. Here, we show that HMGA1 negatively regulates NUMB expression at transcriptional level, by binding its promoter and counteracting c/EBP-ß and at posttranscriptional level, by regulating the expression of MSI1 and of miR-146a. Finally, we report that HMGA1 knockdown-induced NUMB upregulation leads to the downregulation of the NOTCH1 pathway. Therefore, the data reported here indicate that HMGA1 negatively regulates NUMB expression in BTSCs, further supporting HMGA1 targeting as innovative and effective anti-cancer therapy.

HMGA1 silencing reduces stemness and temozolomide resistance in glioblastoma stem cells.

OBJECTIVE: Glioblastoma multiforme (GBM) develops from a small subpopulation of stem-like cells, which are endowed with the ability to self-renew, proliferate and give rise to progeny of multiple neuroepithelial lineages. These cells are resistant to conventional chemo- and radiotherapy and are hence also responsible for tumor recurrence. HMGA1 overexpression has been shown to correlate with proliferation, invasion, and angiogenesis of GBMs and to affect self-renewal of cancer stem cells from colon cancer. The role of HMGA1 in GBM tumor stem cells is not completely understood. RESEARCH DESIGN AND METHODS: We have investigated the role of HMGA1 in brain tumor stem cell (BTSC) self-renewal, stemness and resistance to temozolomide by shRNA- mediated HMGA1 silencing. RESULTS: We first report that HMGA1 is overexpressed in a subset of BTSC lines from human GBMs. Then, we show that HMGA1 knockdown reduces self-renewal, sphere forming efficiency and stemness, and sensitizes BTSCs to temozolomide. Interestingly, HMGA1 silencing also leads to reduced tumor initiation ability in vivo. CONCLUSIONS: These results demonstrate a pivotal role of HMGA1 in cancer stem cell gliomagenesis and endorse HMGA1 as a suitable target for CSC-specific GBM therapy.

miR-142-3p down-regulation contributes to thyroid follicular tumorigenesis by targeting ASH1L and MLL1.

CONTEXT: A previous micro-RNA expression profile of thyroid follicular adenomas identified miR-142 precursor among the miRNAs downregulated in the neoplastic tissues compared to normal thyroid gland. OBJECTIVE: The aim of this work has been to assess the expression of miR-142-3p in a large panel of follicular thyroid adenomas and carcinomas and evaluate its effect on thyroid cell proliferation and target expression. DESIGN: The expression of miR-142-3p was analyzed by qRT-PCR in thyroid follicular adenomas and carcinomas, compared to normal thyroids. MiR-142-3p expression was restored in WRO cells and the effects on cell proliferation and target expression were evaluated. RESULTS: Here we show that miR-142-3p is downregulated in FTAs, FTCs, and FVPTCs. MiR-142-3p was demonstrated to reduce the proliferation rate of WRO and FTC133 cells, supporting its tumor suppressor role in thyroid cancerogenesis. Moreover, this microRNA was able to downregulate the expression of ASH1L and MLL1, by direct and indirect mechanisms, respectively. Consistently, an inverse correlation between miR-142-3p expression and ASH1L and MLL1 proteins was found in thyroid follicular adenomas and carcinomas. ASH1L and MLL1, which belong to the Trithorax group (TrxG) proteins and are major regulators of Homeobox gene expression, maintain active target gene transcription by histone 3 lysine 4 methylation. Interestingly, we found that FTCs and FTC cell lines express tumor specific, shorter forms of the two proteins. The capability of miR-142-3p to modulate the levels of these tumor-associated forms and to reactivate thyroid-specific Hox gene expression, likely contributes to its tumor suppressive function. CONCLUSIONS: These data demonstrate that miR-142-3p downregulation has a role in thyroid tumorigenesis, by regulating ASH1L and MLL1.

CBX7 gene expression plays a negative role in adipocyte cell growth and differentiation.

We have recently generated knockout mice for the Cbx7 gene, coding for a polycomb group protein that is downregulated in human malignant neoplasias. These mice develop liver and lung adenomas and carcinomas, which confirms a tumour suppressor role for CBX7. The CBX7 ability to downregulate CCNE1 expression likely accounts for the phenotype of the Cbx7-null mice. Unexpectedly, Cbx7-knockout mice had a higher fat tissue mass than wild-type, suggesting a role of CBX7 in adipogenesis. Consistently, we demonstrate that Cbx7-null mouse embryonic fibroblasts go towards adipocyte differentiation more efficiently than their wild-type counterparts, and this effect is Cbx7 dose-dependent. Similar results were obtained when Cbx7-null embryonic stem cells were induced to differentiate into adipocytes. Conversely, mouse embryonic fibroblasts and human adipose-derived stem cells overexpressing CBX7 show an opposite behaviour. These findings support a negative role of CBX7 in the control of adipocyte cell growth and differentiation.

HMGA1 silencing restores normal stem cell characteristics in colon cancer stem cells by increasing p53 levels.

High-mobility group A1 (HMGA1) proteins are architectural chromatinic proteins, abundantly expressed during embryogenesis and in most cancer tissues, but expressed at low levels or absent in normal adult tissues. Several studies have demonstrated that HMGA1 proteins play a causal role in neoplastic cell transformation. The aim of this study was to investigate the role of these proteins in the control of cancer stem cells (CSCs), which have emerged as a preferred target in cancer therapy, because of their role in cancer recurrence. We observed that HMGA1 is overexpressed in colon tumour stem cell (CTSC) lines compared to normal and colon cancer tissues. We demonstrated that HMGA1 silencing in CTSCs increases stem cell quiescence and reduces self-renewal and sphere-forming efficiency (SFE). The latter, together with the upregulation and asymmetric distribution of NUMB, is indicative of the recovery of an asymmetric division pattern, typical of normal stem cells. We further found that HMGA1 transcriptionally regulates p53, which is known to control the balance between symmetric and asymmetric divisions in CSCs. Therefore, our data indicate a critical role for HMGA1 in regulating both self-renewal and the symmetric/asymmetric division ratio in CSCs, suggesting that blocking HMGA1 function may be an effective anti-cancer therapy.

Let-7a down-regulation plays a role in thyroid neoplasias of follicular histotype affecting cell adhesion and migration through its ability to target the FXYD5 (Dysadherin) gene.

CONTEXT: Thyroid neoplasias of the follicular histotype include the benign follicular adenomas and the malignant follicular carcinomas. Although several genetic lesions have already been described in human thyroid follicular neoplasias, the mechanisms underlying their development are still far from being completely elucidated. MicroRNAs (miRs or miRNAs) have recently emerged as important regulators of gene expression, also playing a key role in the process of carcinogenesis. OBJECTIVE: The aim of our work has been to identify the miRNAs differentially expressed in human thyroid follicular neoplasias and define their role in thyroid carcinogenesis. DESIGN: The miRNA expression profile of 10 human thyroid follicular adenomas was compared to that of 10 normal thyroid tissues. RESULTS: The miRNA expression profiles revealed the down-regulation of let-7a in thyroid follicular adenomas compared to normal thyroid. Then, quantitative RT-PCR analyses validated the microarray data and showed a significantly higher decrease in let-7a expression in follicular carcinomas. Enforced let-7a expression in the follicular thyroid carcinoma cell line WRO induces an epithelial-like phenotype, increases cell adhesion, and decreases cell migration. Conversely, silencing of let-7a in the normal rat thyroid cell line PC Cl 3 has opposite effects. We identified dysadherin (FXYD5), a cell membrane glycoprotein, correlated with tumor progression and invasiveness, as a target of let-7a. Consistently, an inverse correlation between dysadherin and let-7a expression levels was found in human thyroid follicular adenomas and carcinomas. CONCLUSIONS: These results suggest a role of let-7a down-regulation in the development of thyroid neoplasias of the follicular histotype, likely regulating dysadherin protein expression levels.

miR-191 down-regulation plays a role in thyroid follicular tumors through CDK6 targeting.

CONTEXT: Well-differentiated thyroid carcinomas include papillary (PTC) and follicular (FTC) carcinomas. FTC is usually a more aggressive form of cancer than the more common papillary type. miR-191 expression is frequently altered in several neoplasias, being up-regulated in some cases, such as pancreatic carcinomas, and down-regulated in other carcinomas, such as melanomas. OBJECTIVE: The objective was to evaluate the expression and the role of miR-191 in thyroid carcinogenesis. DESIGN: The expression of miR-191 was analyzed in tissues from patients with follicular adenoma (n = 24), FTC (n = 24), PTC (n = 15), anaplastic thyroid carcinoma (n = 8), and the follicular variant of PTC (n = 6) compared with normal thyroid tissues by quantitative RT-PCR. miR-191 expression was restored in the follicular thyroid cell line WRO, and the effects on cell proliferation, migration, and target expression were evaluated. RESULTS: miR-191 is down-regulated in follicular adenoma, FTC, and follicular variant of PTC. We identified CDK6, a serine-threonine kinase involved in the control of cell cycle progression, as a novel target of miR-191. Restoration of miR-191 expression in WRO cells reduces cell growth and migration rate on vitronectin. CDK6 overexpression, correlated with miR-191 down-regulation, was found in follicular adenoma and FTC, suggesting a role of miR-191 down-regulation in the generation of these neoplasias. CONCLUSIONS: Our results suggest that miR-191 down-regulation plays a role in thyroid neoplasias of the follicular histotype, likely by targeting CDK6.

MiR-1 is a tumor suppressor in thyroid carcinogenesis targeting CCND2, CXCR4, and SDF-1alpha.

CONTEXT: Micro-RNA have emerged as an important class of short endogenous RNA that act as posttranscriptional regulators of gene expression and are constantly deregulated in human cancer. MiR-1 has been found down-regulated in lung, colon, and prostate cancer. OBJECTIVES: In this study, we investigated the possible role of miR-1 in thyroid carcinogenesis. DESIGN: We have analyzed miR-1 expression in a panel of thyroid neoplasias including benign and malignant lesions and searched for miR-1 targets. RESULTS: Our results show that miR-1 expression is drastically down-regulated in thyroid adenomas and carcinomas in comparison with normal thyroid tissue. Interestingly, miR-1 down-regulation was also found in thyroid hyperproliferative nonneoplastic lesions such as goiters. We identified the CCND2, coding for the cyclin D2 (CCND2) protein that favors the G1/S transition, CXCR4, and SDF-1α genes, coding for the receptor for the stromal cell derived factor-1 (SDF-1)/CXCL12 chemokine and its ligand SDF-1/CXCL12, respectively, as miR-1 targets. An inverse correlation was found between miR-1 expression and CXC chemokine receptor 4 (CXCR4) and SDF-1α protein levels in papillary and anaplastic thyroid carcinomas. Consistent with a role of the CCND2 protein in cell proliferation and CXCR4 and SDF-1α proteins in cell invasion and metastasis, functional studies demonstrate that miR-1 is able to inhibit thyroid carcinoma cell proliferation and migration. CONCLUSIONS: These results indicate the involvement of miR-1 in thyroid cell proliferation and migration, validating a role of miR-1 down-regulation in thyroid carcinogenesis.