Mario Taffurelli

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.

p66Shc/Notch-3 interplay controls self-renewal and hypoxia survival in human stem/progenitor cells of the mammary gland expanded in vitro as mammospheres.

The comprehension of the basic biology of stem cells is expected to provide a useful insight into the pathogenesis of cancer. In particular, there is evidence that hypoxia promotes stem cell renewal in vitro as well as in vivo. It therefore seems reasonable that stem cell survival and hypoxia response are strictly connected at molecular level. We here report that the 66‐kDa isoform of the SHC gene (p66Shc) is induced in a breast cancer cell line by the exposure to hypoxic environment and that it controls the expression of the stem cell regulatory gene Notch‐3. Then, we show that p66Shc/Notch‐3 interplay modulates self‐renewal (by inducing the Notch‐ligand Jagged‐1) and hypoxia survival (by inducing the hypoxia‐survival gene carbonic anhydrase IX) in mammary gland stem/progenitor cells, expanded in vitro as multicellular spheroids (mammospheres). We conclude that mechanisms that regulate stem cell renewal and hypoxia survival are integrated at the level of the p66Shc/Notch3 interplay. Because Notch‐3, Jagged‐1, and carbonic anhydrase IX are dysregulated in breast cancer, and because p66Shc is an aging‐regulating gene, we envision that these data may help in understanding the relationship among aging, cancer, and stem 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

TNFalpha up-regulates SLUG via the NF-kappaB/HIF1alpha axis, which imparts breast cancer cells with a stem cell-like phenotype

Extracellular and intracellular mediators of inflammation, such as tumor necrosis factor alpha (TNFα) and NF‐kappaB (NF‐κB), play major roles in breast cancer pathogenesis, progression and relapse. SLUG, a mediator of the epithelial–mesenchymal transition process, is over‐expressed in CD44+/CD24− tumor initiating breast cancer cells and in basal‐like carcinoma, a subtype of aggressive breast cancer endowed with a stem cell‐like gene expression profile. Cancer stem cells also over‐express members of the pro‐inflammatory NF‐κB network, but their functional relationship with SLUG expression in breast cancer cells remains unclear. Here, we show that TNFα treatment of human breast cancer cells up‐regulates SLUG with a dependency on canonical NF‐κB/HIF1α signaling, which is strongly enhanced by p53 inactivation. Moreover, SLUG up‐regulation engenders breast cancer cells with stem cell‐like properties including enhanced expression of CD44 and Jagged‐1 in conjunction with estrogen receptor alpha down‐regulation, growth as mammospheres, and extracellular matrix invasiveness. Our results reveal a molecular mechanism whereby TNFα, a major pro‐inflammatory cytokine, imparts breast cancer cells with stem cell‐like features, which are connected to increased tumor aggressiveness. J. Cell. Physiol. 225: 682–691, 2010.

Neoplastic involvement of nipple-areolar complex in invasive breast cancer

The neoplastic involvement of the nipple-areolar complex was histologically studied in 1,291 available consecutive mastectomy specimens with primary invasive breast carcinoma. Tumor involvement of the nipple-areolar complex was found in 150 specimens (12 percent) and was not suspected on gross examination in 99 patients (8 percent). A significant finding of our study was the relatively high rate of tumor foci in the nipple-areolar complex (7 percent) in those patients with early invasive stage I or II breast carcinoma eligible for conservative therapy. Analysis of nipple-areolar complex involvement with consideration of different clinico-morphologic variables indicates that it was directly associated with tumor size. No significant correlation was found with axillary metastases, tumor histologic type, or with the presence of noninvasive cancer in the vicinity of the dominant tumor. Our estimate of the significant change of finding tumor in the nipple-areolar complex, especially in the patient group eligible for conservative therapy, underlines the need for postoperative radiation.

DIFFERENTIATION PATHWAYS IN PRIMARY INVASIVE BREAST CARCINOMA AS SUGGESTED BY INTERMEDIATE FILAMENT AND BIOPATHOLOGICAL MARKER EXPRESSION

The expression of intermediate filament proteins (IFPs) in 65 primary breast carcinomas was analysed by a panel of specific antibodies. Results were integrated with the oestrogen and progesterone receptor (ER and PGR) status, Ki‐67 marking, and epidermal growth factor receptor (EGFr) expression. Invasive breast carcinomas could be divided into three main groups: group 1 revealed positivity only for ‘simple epithelial’ cytokeratins (CKs 7, 8, 18, and 19); group 2 also stained with the antibodies K8.12 and 34βE12; while group 3 showed co‐expression of CKs 14 and 17, vimentin, and α‐smooth muscle actin. Group 3 consistently comprised tumours with the highest Ki‐67 levels, EGFr positivity, and ER‐PGR negative status. On the other hand, groups 1 and 2 usually exhibited a positive hormonal status, lower proliferative activity, and EGFr negativity. The results of this study indicate that the determination of IFPs can significantly contribute to the identification of groups of patients with different biopathological settings and possibly different clinical behaviour.

Novel Dyskerin-Mediated Mechanism of p53 Inactivation through Defective mRNA Translation

In up to 60% of human cancers, p53 gene mutations are responsible for direct inactivation of the tumor suppressor function of p53. Alternative mechanisms of p53 inactivation described thus far mainly affect its posttranslational regulation. In X-linked dyskeratosis congenita, a multisystemic syndrome characterized by increased cancer susceptibility, mutations of the DKC1 gene encoding dyskerin cause a selective defect in the translation of a subgroup of internal ribosome entry site (IRES)-containing cellular mRNAs. In this study, we show that impairment of dyskerin function can cause p53 inactivation due to a defect in p53 mRNA translation. siRNA-mediated reduction of dyskerin levels caused a decrease of p53 mRNA translation, protein levels, and functional activity, both in human breast cancer cells and in primary mammary epithelial progenitor cells. These effects seemed to be independent of the known role of dyskerin in telomerase function, and they were associated with a specific impairment of translation initiation mediated by IRES elements present in p53 mRNA. In a series of human primary breast cancers retaining wild-type p53, we found that low levels of dyskerin expression were associated with reduced expression of p53-positive target genes. Our findings suggest that a dyskerin-mediated mechanism of p53 inactivation may occur in a subset of human tumors.

c-erbB-2 over-expression in amplified and non-amplified breast carcinoma samples

We investigated c‐erbB‐2 oncogene amplification and over‐expression in 79 invasive breast carcinoma samples using fluorescence in situ hybridization (FISH) and immuno‐histochemistry, with the aim of studying relationships between neoplasms over‐expressing c‐erbB‐2 with or without amplification and bio‐pathological parameters used in clinical breast cancer. Nineteen samples showed amplification, and all of these were positive by immuno‐histochemistry. Moderate or intense immunostaining was present in a further 22 samples without c‐erbB‐2 amplification and was not related to any increased number of c‐erbB‐2 signals: 15 samples exhibited chromosome 17 polysomy, 3 monosomy and 4 no FISH abnormalities. Thirty‐eight samples were immunonegative: 18 exhibited chromosome 17 polysomy, 9 monosomy and 11 no alterations. Samples having c‐erbB‐2 over‐expression associated with amplification showed DNA aneuploidy and hormonal receptor loss to a greater extent than those expressing c‐erbB‐2 without amplification or immunonegative samples (χ2 test, p = 0.007, 0.008 and 0.008, respectively). The proliferation rate, detected by Ag‐NOR staining, was highest in amplified samples (Kruskal Wallis test, p = 0.009). Our results indicate that tumours showing both c‐erbB‐2 over‐expression and amplification exhibit more aggressive biological characteristics than those with only over‐expression or immunonegative tumours. Since both c‐erbB‐2 amplification and over‐expression have been related to negative responses to chemotherapy and poor prognosis, these differences might have clinical implications. The combination of FISH and immuno‐histochemistry may be helpful to achieve this aim. Int. J. Cancer (Pred. Oncol.) 84:273–277, 1999.

Dyskerin expression influences the level of ribosomal RNA pseudo-uridylation and telomerase RNA component in human breast cancer

Dyskerin is a nucleolar protein, altered in dyskeratosis congenita, which carries out two separate functions, both fundamental for proliferating cells. One function is the pseudo‐uridylation of ribosomal RNA (rRNA) molecules, necessary for their processing, and the other is the stabilization of the telomerase RNA component, necessary for telomerase activity. A significant feature of dyskeratosis congenita is an increased susceptibility to cancer; so far, however, no data have been reported on dyskerin changes in human tumours. Therefore, in this study, the distribution of dyskerin in a large series of human tumours from the lung, breast, and colon, as well as from B‐cell lymphomas, was analysed by immunohistochemistry. Dyskerin proved never to be lost or delocalized outside the nucleolus. A quantitative analysis of dyskerin mRNA expression was then performed in 70 breast carcinomas together with the evaluation of telomerase RNA component levels and rRNA pseudo‐uridylation. Dyskerin mRNA levels were highly variable and directly associated with both telomerase RNA component levels and rRNA pseudo‐uridylation. Dyskerin gene silencing in the MCF‐7 human breast carcinoma cell line reduced telomerase activity and rRNA pseudo‐uridylation. Significantly, patients with low dyskerin expression were characterized by a better clinical outcome than those with a high dyskerin level. These data indicate that dyskerin is not lost in human cancers and that the levels of its expression and function are associated with tumour progression. Copyright

Lichtenstein repair of inguinal hernia with Surgisis inguinal hernia matrix soft-tissue graft in immunodepressed patients

While polypropylene mesh remains the preferred prosthesis material for hernioplasties, there are some problems with infections, intestinal obstruction and fistulization, and migration particularly in immunodepressed patients. A new degradable and reabsorbable material, the porcine small intestinal submucosa (Surgisis) has been developed for hernia repairs in humans. This prospective study evaluated the safety and efficacy of Lichtenstein hernioplasty using the Surgisis inguinal hernia matrix soft-tissue graft as a mesh in ten immunodepressed subjects. Six subjects were HIV-positive in the immunodepressive phase, and the other four had undergone transplantation (three kidney, one liver). There were no intraoperative or postoperative complications, recurrences, or wound infections. Thus Lichtenstein’s hernioplasty using the Surgisis inguinal hernia matrix soft-tissue graft in immunodepressed patients promises safety and efficacy.