Paola Paterini

IL-6 triggers malignant features in mammospheres from human ductal breast carcinoma and normal mammary gland

High serum levels of IL-6 correlate with poor outcome in breast cancer patients. However, no data are available on the relationship between IL-6 and mammary stem/progenitor cells, which may fuel the genesis of breast cancer in vivo. Herein, we address this issue in the MCF-7 breast cancer cell line and in primary human mammospheres (MS), multicellular structures enriched in stem/progenitor cells of the mammary gland. MS from node invasive breast carcinoma tissues expressed IL-6 mRNA at higher levels than did MS from matched non-neoplastic mammary glands. In addition, IL-6 mRNA was detected only in basal-like breast carcinoma tissues, an aggressive breast carcinoma variant showing stem cell features. IL-6 treatment triggered Notch-3-dependent upregulation of the Notch ligand Jagged-1 and promotion of MS and MCF-7-derived spheroid growth. Moreover, IL-6 induced Notch-3-dependent upregulation of the carbonic anhydrase IX gene and promoted a hypoxia-resistant/invasive phenotype in MCF-7 cells and MS. Finally, autocrine IL-6 signaling relied upon Notch-3 activity to sustain the aggressive features of MCF-7-derived hypoxia-selected cells. In conclusion, these data support the hypothesis that IL-6 induces malignant features in Notch-3-expressing stem/progenitor cells from human ductal breast carcinoma and normal mammary gland.

MiR-101 downregulation is involved in cyclooxygenase-2 overexpression in human colon cancer cells.

Overexpressed cyclooxygenase-2 (COX-2) strongly contributes to the growth and invasiveness of tumoral cells in patients affected by colorectal cancer (CRC). It has been demonstrated that COX-2 overexpression depends on different cellular pathways involving both transcriptional and post-transcriptional regulations. We assumed that COX-2 expression could be regulated also by microRNAs (miRNAs) since these short RNA molecules participate to the fine regulation of several genes implicated in cell growth and differentiation. In this paper, we report the inverse correlation between COX-2 and miR-101 expression in colon cancer cell lines and we demonstrated in vitro the direct inhibition of COX-2 mRNA translation mediated by miR-101. Moreover, this correlation was supported by data collected ex vivo, in which colon cancer tissues and liver metastases derived from CRC patients were analyzed. These findings provide a novel molecular insight in the modulation of COX-2 at post-transcriptional level by miR-101 and strengthen the observation that miRNAs are highly implicated in the control of gene expression. An impairment of miR-101 levels could represent one of the leading causes of COX-2 overexpression in colon cancer cells.

The basal-like breast carcinoma phenotype is regulated by SLUG gene expression.

Basal‐like breast carcinoma is an aggressive form of breast cancer, characterized by the absence of oestrogen receptor and HER2 expression, the presence of cytokeratin 5 and epidermal growth factor receptor expression, and by the up‐regulation of stem cell regulatory genes. We show here that tumour tissues expressing high levels of SLUG mRNA show a basal‐like breast carcinoma phenotype and that such tumours also express high levels of stem cell‐regulatory genes, ie CD133, Bmi1. Further, we show that stem/progenitor cells, isolated from ductal breast carcinoma and from normal mammary gland as mammospheres, express SLUG, CD133, and Bmi1 mRNA and show a phenotype similar to that of basal‐like breast carcinoma. We also report that SLUG expression in tumour tissues correlates with that of the hypoxia survival gene carbonic anhydrase IX. In this regard, we report that the exposure of SLUG‐negative/luminal‐like MCF‐7 cells to a hypoxic environment promotes the onset of the basal‐like breast carcinoma phenotype, together with up‐regulation of the SLUG gene, which in turn blunts oestrogen receptor‐α and boosts carbonic anhydrase IX gene expression. Finally, we show that SLUG expression promotes the invasiveness of MCF‐7 cells exposed to hypoxia and sustains the in vivo aggressiveness of hypoxia‐selected, MCF‐7‐derived cells in xenografts. These data indicate that SLUG gene expression is part of a hypoxia‐induced genetic programme which sets up a basal/stem cell‐like, aggressive phenotype in breast cancer cells. Copyright

Insulin-like growth factor 1 receptor expression in wild-type GISTs: A potential novel therapeutic target

Aberrations of the Insulin‐like Growth Factor (IGF) system have been found in association with a variety of cancer types. The potential role of IGF1R has been postulated in a small subset of GISTs, but until now the implications of its aberrations have not been defined. The aim of the study was to examine the IGF1R status in patients with gastric GIST in regard to KIT and PDGFRA genotype. Fresh resection specimens were collected from 8 primary tumours [2 wild‐type (WT) and 6 mutant cases]. IGF1R was studied as gene expression profiling with Affymetrix GeneChip HG‐U133Plus 2.0 arrays and as genomic copy number with SNP array analysis Affymetrix Genome Wide Human SNP 6.0 arrays, and at protein level with western blotting (WB) and immunohistochemistry (IHC). The unsupervised analysis of gene expression profiling of our patients merged with a data set from gastric GISTs identified 2 patients out of 8 with different expression of IGF1R. The data were confirmed by WB and IHC. In particular, IGF1R was upregulated in 2 young patients (<30‐years old), who had both WT disease and metastases at diagnosis. The SNP array analysis showed that none of the tumours had IGF1R amplification. GISTs are characterized by abnormalities of the KIT and PDGFRA receptors that affect prognosis and response to tyrosine kinase inhibitors. Both young adult with WT GIST had the over‐expression of IGF1R at mRNA and protein level. These results further confirm the hypothesis that IGF1R may be a potential therapeutic target in GISTs lacking KIT and PDGFRA mutations.

High Thymidylate Synthase Expression in Colorectal Cancer with Microsatellite Instability: Implications for Chemotherapeutic Strategies

Colon cancers displaying microsatellite instability (MSI) are clinically less aggressive. Based on in vitro studies and recent clinical data, cancers displaying MSI do not respond to 5-fluorouracil (5-FU). The reasons why MSI tumors are clinically less aggressive and do not respond to 5-FU–based therapies have not been fully elucidated. Purpose: We investigated biomolecular markers in an attempt to explain the different clinical behavior and chemotherapeutic responses of MSI and non-MSI colon cancers. Experimental Design: One hundred ninety-two sporadic colon cancers were tested for MSI with five mononucleotide markers and methylation of the hMLH1 promoter. Slides were stained for thymidylate synthase (TS), p53, MDM2, p21WAF1/CIP1, β-catenin, vascular endothelial growth factor, hMLH1, hMSH2, and hMSH6. Tumors were regarded as having wild-type, functional p53 (Fp53) if reduced expression of p53 and positive MDM2 and p21WAF1/CIP1 expressions were found. Results: Of the cases, 12.5% were MSI-H (at least two markers mutated). Of MSI-H cases, 83.3% were characterized by a complete loss of at least one of the mismatch repair proteins, in particular loss of hMLH1 by promoter hypermethylation. MSI-H colon cancers showed higher expression of TS compared with MSS (no mutated markers)/MSI-L (one mutated marker) colon cancers (66.6% for MSI-H versus 14.8% MSS/MSI-L; P < 0.0001); 20.8% of MSI-H cases showed high expression of the vascular endothelial growth factor, compared with 45.8% MSS/MSI-L colon cancers (P = 0.0005); 45.8% MSI-H cases had Fp53 compared 11.9% MSS/MSI-L cases (P < 0.0001). Conclusions: About 12% of colon cancers display MSI mostly due to lack of hMLH1 resulting from promoter hypermethylation. These tumors have high expression of TS and retain fully functional p53 system. Thus, these data suggest why sporadic hMLH1-defective colon cancers often do not respond to 5-FU.

Analysis of all subunits, SDHA, SDHB, SDHC, SDHD, of the succinate dehydrogenase complex in KIT/PDGFRA wild-type GIST.

Mutations of genes encoding the subunits of the succinate dehydrogenase (SDH) complex were described in KIT/PDGFRA wild-type GIST separately in different reports. In this study, we simultaneously sequenced the genome of all subunits, SDHA, SDHB, SDHC, and SDHD in a larger series of KIT/PDGFRA wild-type GIST in order to evaluate the frequency of the mutations and explore their biological role. SDHA, SDHB, SDHC, and SDHD were sequenced on the available samples obtained from 34 KIT/PDGFRA wild-type GISTs. Of these, in 10 cases, both tumor and peripheral blood (PB) were available, in 19 cases only tumor, and in 5 cases only PB. Overall, 9 of the 34 patients with KIT/PDGFRA wild-type GIST carried mutations in one of the four subunits of the SDH complex (six patients in SDHA, two in SDHB, one in SDHC). WB and immunohistochemistry analysis showed that patients with KIT/PDGFRA wild-type GIST who harbored SDHA mutations exhibited a significant downregulation of both SDHA and SDHB protein expression, with respect to the other GIST lacking SDH mutations and to KIT/PDGFRA-mutated GIST. Clinically, four out of six patients with SDHA mutations presented with metastatic disease at diagnosis with a very slow, indolent course. Patients with KIT/PDGFRA wild-type GIST may harbor germline and/or de novo mutations of SDH complex with prevalence for mutations within SDHA, which is associated with a downregulation of SDHA and SDHB protein expression. The presence of germline mutations may suggest that these patients should be followed up for the risk of development of other cancers.

Gene expression profiling of liver metastases from colorectal cancer as potential basis for treatment choice.

At present no reports on gene expression profiling of liver metastases from colorectal cancer are available. We identified two different signatures using Affymetrix platform: epidermal growth factor receptor pathway was upregulated in metachronous lesions, whereas the pathway mainly related to angiogenesis was in synchronous lesions. Synchronous or metachronous liver metastases could be treated differently on the basis of different molecular pathways.

Cyclooxygenase-2/carbonic anhydrase-IX up-regulation promotes invasive potential and hypoxia survival in colorectal cancer cells.

Inflammation promotes colorectal carcinogenesis. Tumour growth often generates a hypoxic environment in the inner tumour mass. We here report that, in colon cancer cells, the expression of the pro‐inflammatory enzyme cyclooxygenase‐2 (COX‐2) associates with that of the hypoxia response gene carbonic anhydrase‐IX (CA‐IX). The COX‐2 knockdown, achieved by the stable infection of a COX‐2 specific short harpin RNA interference (shCOX‐2), down‐regulates CA‐IX gene expression. In colorectal cancer (CRC) cells, PGE2, the main COX‐2 gene products, promotes CA‐IX gene expression by ERK1/2 activation. In normoxic environment, shCOX‐2 infected/CA‐IX siRNA transfected CRC cells show a reduced level of active metalloproteinase‐2 (MMP‐2) that associates with a decreased extracellular matrix invasion capacity. In presence of hypoxia, COX‐2 gene expression and PGE2 production increase. The knockdown of COX‐2/CA‐IX blunts the survival capability of CRC cells in hypoxia. At a high cell density, a culture condition that creates a mild pericellular hypoxic environment, the expression of COX‐2/CA‐IX genes is increased and triggers the invasive potential of colon cancer cells. In human colon cancer tissues, COX‐2/CA‐IX protein expression levels, assessed by Western blot and immunohistochemistry, correlate each other and increase with tumour stage. In conclusion, these data indicate that COX‐2/CA‐IX interplay promotes the aggressive behaviour of CRC cells.

Expression of IGF-1 receptor in KIT/PDGF receptor-α wild-type gastrointestinal stromal tumors with succinate dehydrogenase complex dysfunction

KIT/PDGF receptor-α (PDGFRA) wild-type (WT) gastrointestinal stromal tumors (GIST) are characterized by an overexpression of IGF-1 receptor (IGF1R) at the mRNA and protein level. More recently, germline and somatic mutations in succinate dehydrogenase (SDH) subunits A, B and C have been identified in KIT/PDGFRA WT sporadic GIST. Until now, the molecular basis of IGF1R overexpression in KIT/PDGFRA WT GIST has not been explained. In this brief report we investigate the status of the SDH complex at the genomic and protein level in relation to IGF1R expression at the mRNA and protein level in seven KIT/PDGFRA WT sporadic GIST patients. We found that IGF1R was upregulated in all patients harboring SDH mutations or displaying a SDH dysfunction, with respect to KIT/PDGFRA WT GIST without SDH mutations. Western blot analysis confirmed that all patients with an upregulation of IGF1R mRNA had detectable IGF1R protein expression. This report would suggest that IGF1R overexpression in KIT/PDGFRA WT GIST could be driven by the loss-of-function of the SDH mitochondrial complex.

Differential expression of neural markers in KIT and PDGFRA wild-type gastrointestinal stromal tumours

Pantaleo M A, Astolfi A, Nannini M, Ceccarelli C, Formica S, Santini D, Heinrich M C, Corless C, Dei Tos A P, Paterini P, Catena F, Maleddu A, Saponara M, Di Battista M & Biasco G 
(2011) Histopathology 59, 1071–1080 
Differential expression of neural markers in KIT and PDGFRA wild‐type gastrointestinal stromal tumours