Rita Falcioni

p53 Mediates the Accelerated Onset of Senescence of Endothelial Progenitor Cells in Diabetes

Adverse metabolic factors, including oxidized small and dense low density lipoprotein (ox-dmLDL) can contribute to the reduced number and the impaired functions of circulating endothelial progenitors (EPC) in diabetic patients. To elucidate the molecular mechanisms involved, EPC from normal donors were cultured in the presence of ox-dmLDL. Under these experimental conditions EPC undergo to senescent-like growth arrest. This effect is associated with Akt activation, p21 expression, p53 accumulation, and retinoblastoma protein dephosphorylation and with a reduced protective effect against oxidative damage. Moreover, depletion of endogenous p53 expression by small interfering RNA demonstrates that the integrity of this pathway is essential for senescence to occur. Activation of the Akt/p53/p21 signaling pathway and accelerated onset of senescence are also detectable in EPC from diabetic patients. Finally, diabetic EPC depleted of endogenous p53 do not undergo to senescence-growth arrest and acquire the ability to form tube-like structures in vitro. These observations identify the activation of the p53 signaling pathway as a crucial event that can contribute to the impaired neovascularization in diabetes.

Resensitization of breast cancer cells to anoikis by Tropomyosin-1: Role of Rho kinase-dependent cytoskeleton and adhesion

Two most common properties of malignant cells are the presence of aberrant actin cytoskeleton and resistance to anoikis. Suppression of several key cytoskeletal proteins, including tropomyosin-1 (TM1), during neoplastic transformation is hypothesized to contribute to the altered cytoskeleton and neoplastic phenotype. Using TM1 as a paradigm, we have shown that cytoskeletal proteins induce anoikis in breast cancer (MCF-7 and MDA MB 231) cells. Here, we have tested the hypothesis that TM1-mediated cytoskeletal changes regulate integrin activity and the sensitivity to anoikis. TM1 expression in MDA MB 231 cells promotes the assembly of stress fibers, induces rapid anoikis via caspase-dependent pathways involving the release of cytochrome c. Further, TM1 inhibits binding of MDA MB 231 cells to collagen I, but promotes adhesion to laminin. Inhibition of Rho kinase disrupts TM1-mediated cytoskeletal reorganization and adhesion to the extracellular matrix components, whereas the parental cells attach to collagen I, spread and form extensive actin meshwork in the presence of Rho kinase inhibitor, underscoring the differences in parental and TM1-transduced breast cancer cells. Further, treatment with the cytoskeletal disrupting drugs rescues the cells from TM1-induced anoikis. These new findings demonstrate that the aberrant cytoskeleton contributes to neoplastic transformation by conferring resistance to anoikis. Restoration of stress fiber network through enhanced expression of key cytoskeletal proteins may modulate the activity of focal adhesions and sensitize the neoplastic cells to anoikis.

Activation of p53 function in carcinoma cells by the alpha6beta4 integrin

The interaction of integrins with extracellular matrix is known to promote cell survival by inhibiting apoptotic signaling. In contrast, we demonstrate here that the α6β4 integrin induces apoptosis in carcinoma cells by stimulating p53 function. Specifically, we show that expression of α6β4 in carcinoma cells that lack this integrin stimulates an increase in the transactivating function of p53 as demonstrated by the ability of this integrin to up-regulate the expression of a p53-sensitive reporter gene as well as the endogenous p53 response gene, bax. In addition, we report that α6β4 triggers apoptosis in carcinoma cells that express wild-type but not mutant p53 and that these α6β4 functions are inhibited by a dominant negative p53 construct. Importantly, we provide a link between integrin signaling and p53 activation by demonstrating that the clustering of α6β4 with a β4integrin-specific antibody promotes p53-dependent apoptosis in cells that express both α6β4 and wild-type p53. These studies are the first to demonstrate that a specific integrin can promote apoptosis by activating p53. Moreover, given the ability of α6β4 to stimulate invasion (Shaw, L. M., Rabinovitz, I., Wang, H. F., Toker, A., and Mercurio, A. M. (1997) Cell 91, 949–960), these studies suggest that the ability of α6β4 to promote carcinoma progression will be enhanced in tumor cells that express mutant, inactive forms of p53.

The α6β4 integrin can regulate ErbB-3 expression: Implications for α6β4 signaling and function

The integrin α6β4 has been shown to facilitate key functions of carcinoma cells, including their ability to migrate, invade, and evade apoptosis. The mechanism involved seems to be a profound effect of α6β4 on specific signaling pathways, especially the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. An intimate relationship between α6β4 and growth factor receptors may explain this effect of α6β4 on signaling. Previously, we showed that α6β4 and ErbB-2 can function synergistically to activate the PI3K/Akt pathway. Given that ErbB-2 can activate PI3K only when it heterodimerizes with other members of the epidermal growth factor receptor family, these data imply that other receptors cooperate in this process. Here, we report that α6β4 can regulate the expression of ErbB-3 using several different models and that the consequent formation of an ErbB-2/ErbB-3 heterodimer promotes the α6β4-dependent activation of PI3K/Akt and the ability of this integrin to impede apoptosis of carcinoma cells. Our data also support the hypothesis that α6β4 can regulate ErbB-3 expression at the translational level as evidenced by the findings that α6β4 does not increase ErbB-3 mRNA significantly, and that this regulation is both rapamycin sensitive and dependent on eukaryotic translation initiation factor 4E. These findings provide one mechanism to account for the activation of PI3K by α6β4 and they also provide insight into the regulation of ErbB-3 in carcinoma cells. [Cancer Res 2007;67(4):1645–52]

Induction of ErbB-3 Expression by α6β4 Integrin Contributes to Tamoxifen Resistance in ERβ1-Negative Breast Carcinomas

Background Tamoxifen is still the most widely used drug in hormone therapy for the treatment of breast cancer. Its benefits in adjuvant treatment are well documented in controlled and randomized clinical studies, which have demonstrated an increase in disease-free intervals of patients with positive hormonal receptors. However, the mechanisms involved in endocrine resistance are not clear. Laboratory and clinical data now indicate that bi-directional molecular cross-talk between nuclear or membrane ER and growth factor receptor pathways may be involved in endocrine resistance. We recently found a functional interaction between α6β4 integrin and ErbB-3 receptor to maintain the PI3K/Akt survival pathway of mammary tumour cells. We sought to improve understanding of this process in order to provide the involvement of both receptors insight into mechanism of Tamoxifen resistance. Methods and Findings Using human breast cancer cell lines displaying different levels of α6β4 and ErbB-3 receptors and a series of 232 breast cancer biopsies from patients submitted to adjuvant Tamoxifen monotherapy for five years, we evaluated the functional interaction between both receptors in relationship to Tamoxifen responsiveness. In mammary carcinoma cells, we evidenced that the α6β4 integrin strongly influence Akt phosphorylation through ErbB-3 protein regulation. Moreover, the ErbB-3 inactivation inhibits Akt phosphorylation, induces apoptosis and inhibits in vitro invasion favouring Tamoxifen responsiveness. The analysis of human tumors revealed a significant relationship between α6β4 and ErbB-3 in P-Akt-positive and ERβ1-negative breast cancers derived from patients with lower disease free survival. Conclusions We provided evidence that a strong relationship occurs between α6β4 and ErbB-3 positivity in ERβ1-negative breast cancers. We also found that the association between ErbB-3 and P-Akt positivity mainly occurs in ERβ1-negative breast cancer derived from patients with lower DFS indicating that both receptors are clinically relevant in predicting the response to Tamoxifen.

Purification and Characterization of Adipose-Derived Stem Cells From Patients With Lipoaspirate Transplant

Techniques for medical tissue regeneration require an abundant source of human adult stem cells. There is increasing evidence that adipose stem cells contribute to restoration of tissue vascularization and organ function. The object of our study was to isolate and characterize adult adipose-derived stem cells from patients undergoing on lipoaspirate transplant with the aim to improve tissue regeneration. Adipose-derived stem cells were isolated and purified from the lipoaspirate of 15 patients and characterized for CD markers and the ability to differentiate toward the adipogenic lineage. We found that purified adipose stem cells express high level of CD49d, CD44, CD90, CD105, CD13, and CD71 and these markers of staminality were maintained at high level for at least 3 months and seven passages of in vitro culture. As expected, these cells resulted negative for the endothelial and hematopoietic-specific markers CD31, CD106, CD34, and CD45. Differentiation towards adipogenic lineage demonstrated that purified adipose-derived stem cells are still able to become adipocytes at least 3 months after in vitro culture. The analysis of Akt and MAPK phosphorylation confirmed a modulation of their activity during differentiation. Interestingly, we established for the first time that, among the p53 family members, a strong upregulation of p63 expression occurs in adipocytic differentiation, indicating a role for this transcription factor in adipocytic differentiation. Taken together, these data indicate that purified lipoaspirate-derived stem cells maintain their characteristic of staminality for a long period of in vitro culture, suggesting that they could be applied for cell-based therapy to improve autologous lipoaspirate transplant.

Loss of β4 Integrin Subunit Reduces the Tumorigenicity of MCF7 Mammary Cells and Causes Apoptosis upon Hormone Deprivation

Purpose: The α6β4 integrin, a laminin receptor, has been implicated from many studies in tumor progression and invasion. We showed that the β4 integrin subunit associates with the ErbB-2 tyrosine kinase in human mammary carcinoma cell lines and that its overexpression in NIH3T3/ErbB-2–transformed cells causes a constitutive activation of phosphatidylinositol 3-kinase (PI3K), inducing a strong increase of their invasive capacity. In this study, we investigated the biological consequences of interference with the endogenous β4 integrin subunit expression. Experimental Design:In vitro and in vivo tumor growth and the biochemical consequences of β4 integrin inactivation were studied in mammary tumor cells by using short hairpin RNA approach. Results: Our data show that tumor growth of mammary tumor cells strictly depends on β4 expression, confirming the relevance of β4 protein in these cells. Moreover, interference with β4 expression significantly reduces endogenous PI3K activity and AKT and mammalian target of rapamycin phosphorylation. Accordingly, with these results and considering that PI3K activity in mammary tumor plays a relevant role in hormone resistance, we asked whether β4 expression might be relevant for hormone responsiveness in these cells. Data reported indicate that the interference with endogenous β4 expression, upon hormone deprivation, induces caspase-9 and cytochrome c–mediated apoptosis, which is enhanced upon tamoxifen treatment. On the other hand, the expression of myr-AKT in MCF7 β4–short hairpin RNA cells rescues the cells from apoptosis in the absence of hormones and upon tamoxifen treatment. Conclusions: Overall, these results confirm the relevance of β4 expression in mammary tumors and indicate this integrin as a relevant target for tumor therapy.

Homeodomain-Interacting Protein Kinase-2 Restrains Cytosolic Phospholipase A2^ Dependent Prostaglandin E2 Generation in Human Colorectal Cancer Cells

Purpose: Homeodomain-interacting protein kinase-2 (HIPK2), a corepressor for homeodomain transcription factors, is a multifunctional kinase whose role in tumor cell survival is not completely clarified. We addressed whether HIPK2 restrains colon tumorigenesis by turning off cytosolic phospholipase A2 (cPLA2)-dependent prostaglandin E2 (PGE2) generation in the light of overwhelming evidence suggesting the contribution of this prostanoid in a variety of cancers. Experimental Design: In the human colorectal cancer cell line, RKO, we studied the effect of RNA interference for HIPK2 (HIPK2i) on prostanoid biosynthesis, both in the absence and in the presence of the cPLA2 inhibitor arachidonyl trifluoromethyl ketone. We evaluated the role of HIPK2 in the cPLA2 gene regulation by reverse transcriptase-PCR, transcriptional activity, and chromatin immunoprecipitation analyses. The involvement of HIPK2 in tumorigenicity in vivo was studied by tumor growth of HIPK2i cells in nude mice. We compared the gene expression of HIPK2 and cPLA2 in human colorectal cancer specimens by reverse transcriptase-PCR. Results: HIPK2 silencing was associated with rousing PGE2 biosynthesis that was profoundly suppressed by the cPLA2 inhibitor. HIPK2 overexpression, along with histone deacetylase-1, inhibited the cPLA2-luc promoter that is strongly acetylated in HIPK2i cells. The tumors derived from HIPK2i cells injected in nude mice showed noticeably increased growth compared with parental cells. HIPK2 mRNA levels were significantly higher in colorectal cancers of patients with familial adenomatous polyposis, which showed undetectable cPLA2 levels compared with sporadic colorectal cancer expressing cPLA2. Conclusions: Our findings reveal the novel mechanism of HIPK2 to restrain progression of human colon tumorigenesis, at least in part, by turning off cPLA2-dependent PGE2 generation.

Involvement of α6β4 integrin in the mechanisms that regulate breast cancer progression

Integrin α6β4 is mostly expressed in epithelial tissues and endothelial and Schwann cells. Expression of α6β4 is increased in many epithelial tumours, implicating its involvement in tumour malignancy. Moreover, this integrin activates several key signalling molecules in carcinoma cells, but its ability to activate the phosphatidylinositol 3-kinase/Akt pathway is among the mechanisms by which α6β4 integrin regulates tumour behaviour. In this review we discuss the biological and clinical features of α6β4 integrin that allow it to promote tumour survival and progression of mammary tumours.

Transcription factor NF-Y induces apoptosis in cells expressing wild-type p53 through E2F1 upregulation and p53 activation.

The CCAAT-binding transcription factor NF-Y plays a central role in regulating cellular proliferation by controlling the expression of genes required for cell-cycle progression such as cyclin A, cyclin B1, cyclin B2, cdc25A, cdc25C, and cdk1. Here we show that unrestricted NF-Y activity leads to apoptosis in an E2F1- and wild-type p53 (wtp53)-dependent manner. Unrestricted NF-Y activity induced an increase in E2F1 mRNA and protein levels. Furthermore, NF-Y directly bound the E2F1 promoter and this correlated with the appearance of open chromatin marks. The ability of NF-Y to induce apoptosis was impaired in cells lacking E2F1 and wtp53. Moreover, NF-Y overexpression elicited phosphorylation of wt p53Ser18 in an E2F1-dependent manner. Our findings establish that NF-Y acts upstream of E2F1 in p53-mediated apoptosis.