Hanna M. Romanska

CD151 Regulates Tumorigenesis by Modulating the Communication between Tumor Cells and Endothelium

The tetraspanin CD151 forms stoichiometric complexes with laminin-binding integrins (e.g., α3β1, α6β1, and α6β4) and regulates their ligand-binding and signaling functions. We have found that high expression of CD151 in breast cancers is associated with decreased overall survival (3.44-fold higher risk of death). Five-year estimated survival rates were 45.8% (95% confidence interval, 16.4-71.4%) for CD151-positive patients and 79.9% (95% confidence interval, 62.2-90.0%) for CD151-negative patients. Furthermore, CD151 was positively associated with axillary lymph node involvement. To study the biological significance of this observation, we investigated the contribution of CD151 in breast cancer tumorigenesis using MDA-MB-231 cells as a model system. Stable down-regulation of this tetraspanin by short-hairpin RNA decreased the tumorigenicity of these cells in mice. Detailed immunohistologic analysis of CD151(+) and CD151(−) xenografts showed differences in tumor vascular pattern. Vascularization observed at the subcutaneous border of the CD151(+) tumors was less pronounced or absent in the CD151(−) xenografts. In vitro experiments have established that depletion of CD151 did not affect the inherent proliferative capacity of breast cancer cells in three-dimensional extracellular matrices, but modified their responses to endothelial cells in coculture experiments. The modulatory activity of CD151 was dependent on its association with both α3β1 and α6β4 integrins. These data point to a new role of CD151 in tumorigenesis, whereby it functions as an important regulator of communication between tumor cells and endothelial cells. These results also identify CD151 as a potentially novel prognostic marker and target for therapy in breast cancer. (Mol Cancer Res 2009;7(6):787–98)

Tetraspanin CD151 Regulates Transforming Growth Factor {beta} Signaling: Implication in Tumor Metastasis

Tetraspanin CD151 is associated with laminin-binding integrins and controls tumor cell migration and invasion. By analyzing responses of breast cancer cells to various growth factors, we showed that depletion of CD151 specifically attenuates transforming growth factor beta1 (TGFbeta1)-induced scattering and proliferation of breast cancer cells in three-dimensional Matrigel. CD151-dependent cell scattering requires its association with either alpha3beta1 or alpha6 integrins, but it is independent of the recruitment of CD151 to tetraspanin-enriched microdomains. We also found that CD151 regulates the compartmentalization of TGF-beta type I receptor (TbetaRI/ALK-5) and specifically controls the TGFbeta1-induced activation of p38. In contrast, signaling leading to activation of Smad2/3, c-Akt, and Erk1/2 proteins was comparable in CD151(+) and CD151(-) cells. Attenuation of TGFbeta1-induced responses correlated with reduced retention in the lung vascular bed, inhibition of pneumocyte-induced scattering of breast cancer cells in three-dimensional Matrigel, and decrease in experimental metastasis to the lungs. These results identify CD151 as a positive regulator of TGFbeta1-initiated signaling and highlight the important role played by this tetraspanin in TGFbeta1-induced breast cancer metastasis.

Tetraspanins in human epithelial malignancies

Transmembrane proteins of the tetraspanin superfamily are implicated in a broad spectrum of cellular processes in many biological systems in both health and disease. Tetraspanins form specialized membrane microdomains on the cell surface which control cell proliferation and migration through various adhesion and growth factor receptors. Recent extensive research has shown that expression of various tetraspanins and their associated partners is deregulated in human malignancies. Although, for the most part, the degree of involvement of tetraspanins in carcinogenesis remains to be established, increasing evidence suggests that these proteins might be of clinical significance in at least some malignancies. Here we provide a comprehensive review of the existing data on expression of tetraspanins in breast cancer and other human epithelial tumours, with an emphasis on their value as potential prognostic markers. Copyright

Tetraspanin CD151 regulates growth of mammary epithelial cells in three-dimensional extracellular matrix: implication for mammary ductal carcinoma in situ.

Tetraspanin CD151 is associated with laminin-binding integrins (i.e., alpha(3)beta(1), alpha(6)beta(1), and alpha(6)beta(4)) and regulates tumor cell migration and invasion. Here, we examined the role of CD151 in proliferation of mammary epithelial cells using in vitro and in vivo models. Depletion of CD151 suppressed growth of HB2 cells, a nontumorigenic breast epithelial cell line, in three-dimensional (3D) extracellular matrices (ECM) and in Matrigel-based xenografts. Whereas the presence of alpha(3)beta(1) (but not alpha(6) integrins) was necessary to support growth of HB2 cells in 3D ECM, the pro-proliferative activity of CD151 did not require direct interaction with integrins. Furthermore, depletion of CD151 potentiated formation of the internal lumen and partial restoration of polarity when HB2 cells were cultured in 3D ECM. This correlated with a decrease in phosphorylation levels of extracellular signal-regulated kinase 1/2 and cAkt in CD151-negative cells and increase in activation of caspase-3. Accordingly, the number of CD151-positive colonies with internal lumen was increased by approximately 5-fold when cells were cultured in the presence of MAP/ERK kinase (U0126) and phosphoinositide 3-kinase (LY29004) inhibitors. To establish the physiologic relevance of pro-proliferative and morphogenetic activities of CD151, we analyzed the expression of this tetraspanin in ductal carcinoma in situ (DCIS), which is characterized by neoplastic proliferation of mammary epithelial cells. Strong homogeneous membrane expression of CD151 was found to be associated with a high grade of DCIS (P = 0.004). Taken together, these results strongly suggest that CD151 complexes play a crucial role in the development of hyperproliferative diseases in the mammary gland.

CD151 in cancer progression and metastasis: a complex scenario.

Originally identified as a molecular organizer of interacting proteins into tetraspanin-enriched microdomains, the tetraspanin CD151 has now been shown to be involved in tumour progression. Increasing evidence emerging from in vitro, in vivo and clinical analyses implicates this tetraspanin in supporting growth of various types of tumours at different levels. It affects both cell autonomous behavior and communication with neighboring cells and the microenvironment. CD151 regulates post-adhesion events, that is, cell spreading, migration and invasion including subsequent intravasation and formation of metastasis. Present on both neoplastic and endothelial cells, CD151 is engaged in promotion of tumour neovascularization. The molecular mechanism of CD151 in cancer is based on its ability to organize distribution and function of interacting proteins, ie, laminin-binding integrins (α3β1, α6β1 and α6β4), receptors for growth factors (HGFR, EGFR and TGF-β1R) and matrix metalloproteinases (MMP-7, MMP-2 and MMP-9), which indicates its importance in disease development. Results of clinical analyses of CD151 expression in different types of cancer and a large number of in vivo models demonstrate its impact on tumour growth and invasion and implicate CD151 as a valuable diagnostic and prognostic marker as well as a potential target for anti-cancer therapy.

Integrin α3β1–CD151 complex regulates dimerization of ErbB2 via RhoA

Integrin α3β1 regulates adhesive interactions of cells with laminins and have a critical role in adhesion-dependent cellular responses. Here, we examined the role of α3β1-integrin in ErbB2-dependent proliferation of breast cancer cells in three-dimensional laminin-rich extracellular matrix (3D lr-ECM). Depletion of α3β1 in ErbB2-overexpressing breast cancer cells suppressed growth and restore cell polarity in 3D lr-ECM. The phenotype of α3β1-depleted cells was reproduced upon depletion of tetraspanin CD151 and mirrored that of the cells treated with Herceptin, an established ErbB2 antagonist. Breast cancer cells expressing the α3β1–CD151 complex have higher steady-state phosphorylation of ErbB2 and show enhanced dimerization of the protein when compared with α3β1-/CD151-depleted cells. Furthermore, Herceptin-dependent dephosphorylation of ErbB2 was only observed in α3β1–CD151-expressing cells. Importantly, the inhibitory activity of Herceptin was more pronounced when cells expressed both α3β1 and CD151. We also found that the level of active RhoA was increased in α3β1- and CD151-depleted cells and that Rho controls dimerization of ErbB2. Expression of α3β1 alone did not have significant prognostic value in patients with invasive ductal carcinoma of the breast. However, expression of α3β1 in combination with CD151 represented a more stringent indicator of poor survival than CD151 alone. Taken together, these results demonstrate that the α3β1–CD151 complex has a critical regulatory role in ErbB2-dependent signalling and thereby may be involved in breast cancer progression.

p27Kip1 and p130 Cooperate To Regulate Hematopoietic Cell Proliferation In Vivo

ABSTRACT To investigate the potential functional cooperation between p27Kip1 and p130 in vivo, we generated mice deficient for both p27Kip1 and p130. In p27Kip1−/−; p130−/− mice, the cellularity of the spleens but not the thymi is significantly increased compared with that of their p27Kip1−/− counterparts, affecting the lymphoid, erythroid, and myeloid compartments. In vivo cell proliferation is significantly augmented in the B and T cells, monocytes, macrophages, and erythroid progenitors in the spleens of p27Kip1−/−; p130−/− animals. Immunoprecipitation and immunodepletion studies indicate that p130 can compensate for the absence of p27Kip1 in binding to and repressing CDK2 and is the predominant CDK-inhibitor associated with the inactive CDK2 in the p27Kip1−/− splenocytes. The finding that the p27Kip1−/−; p130−/− splenic B cells are hypersensitive to mitogenic stimulations in vitro lends support to the concept that the hyperproliferation of splenocytes is not a result of the influence of their microenvironment. In summary, our findings provide genetic and molecular evidence to show that p130 is a bona fide cyclin-dependent kinase inhibitor and cooperates with p27Kip1 to regulate hematopoietic cell proliferation in vivo.

Phosphorylation of RSK2 at Tyr529 by FGFR2-p38 enhances human mammary epithelial cells migration

The members of p90 ribosomal S6 kinase (RSK) family of Ser/Thr kinases are downstream effectors of MAPK/ERK pathway that regulate diverse cellular processes including cell growth, proliferation and survival. In carcinogenesis, RSKs are thought to modulate cell motility, invasion and metastasis. Herein, we have studied an involvement of RSKs in FGF2/FGFR2-driven behaviours of mammary epithelial and breast cancer cells. We found that both silencing and inhibiting of FGFR2 attenuated phosphorylation of RSKs, whereas FGFR2 overexpression and/or its stimulation with FGF2 enhanced RSKs activity. Moreover, treatment with ERK, Src and p38 inhibitors revealed that p38 kinase acts as an upstream RSK2 regulator. We demonstrate for the first time that in FGF2/FGFR2 signalling, p38 but not MEK/ERK, indirectly activated RSK2 at Tyr529, which facilitated phosphorylation of its other residues (Thr359/Ser363, Thr573 and Ser380). In contrast to FGF2-triggered signalling, inhibition of p38 in the EGF pathway affected only RSK2-Tyr529, without any impact on the remaining RSK phosphorylation sites. p38-mediated phosphorylation of RSK2-Tyr529 was crucial for the transactivation of residues located at kinase C-terminal domain and linker-region, specifically, in the FGF2/FGFR2 signalling pathway. Furthermore, we show that FGF2 promoted anchorage-independent cell proliferation, formation of focal adhesions and cell migration, which was effectively abolished by treatment with RSKs inhibitor (FMK). These indicate that RSK2 activity is indispensable for FGF2/FGFR2-mediated cellular effects. Our findings identified a new FGF2/FGFR2-p38-RSK2 pathway, which may play a significant role in the pathogenesis and progression of breast cancer and, hence, may present a novel therapeutic target in the treatment of FGFR2-expressing tumours.

Lack of CD151/integrin α3β1 complex is predictive of poor outcome in node-negative lobular breast carcinoma: opposing roles of CD151 in invasive lobular and ductal breast cancers.

Background:The proposed involvement of CD151 in breast cancer (BCa) progression is based on findings from studies in invasive ductal carcinoma (IDC). The IDC and invasive lobular carcinoma (ILC) represent distinct disease entities. Here we evaluated clinical significance of CD151 alone and in association with integrin α3β1 in patients with ILC in context of the data of our recent IDC study.Methods:Expression of CD151 and/or integrin α3β1 was evaluated in ILC samples (N=117) using immunohistochemistry. The findings were analysed in relation to our results from an IDC cohort (N=182) demonstrating a prognostic value of an expression of CD151/integrin α3β1 complex in patients with HER2-negative tumours.Results:Unlike in the IDCs, neither CD151 nor CD151/α3β1 complex showed any correlation with any of the ILC characteristics. Lack of both CD151 and α3β1 was significantly correlated with poor survival (P=0.034) in lymph node-negative ILC N(−) cases. The CD151−/α3β1− patients had 3.12-fold higher risk of death from BCa in comparison with the rest of the ILC N(−) patients.Conclusions:Biological role of CD151/α3β1 varies between ILC and IDC. Assessment of CD151/α3β1 might help to identify ILC N(−) patients with increased risk of distant metastases.

Tetraspanin Tspan9 regulates platelet collagen receptor GPVI lateral diffusion and activation.

Abstract The tetraspanins are a superfamily of four-transmembrane proteins, which regulate the trafficking, lateral diffusion and clustering of the transmembrane proteins with which they interact. We have previously shown that tetraspanin Tspan9 is expressed on platelets. Here we have characterised gene-trap mice lacking Tspan9. The mice were viable with normal platelet numbers and size. Tspan9-deficient platelets were specifically defective in aggregation and secretion induced by the platelet collagen receptor GPVI, despite normal surface GPVI expression levels. A GPVI activation defect was suggested by partially impaired GPVI-induced protein tyrosine phosphorylation. In mechanistic experiments, Tspan9 and GPVI co-immunoprecipitated and co-localised, but super-resolution imaging revealed no defects in collagen-induced GPVI clustering on Tspan9-deficient platelets. However, single particle tracking using total internal reflection fluorescence microscopy showed that GPVI lateral diffusion was reduced by approximately 50% in the absence of Tspan9. Therefore, Tspan9 plays a fine-tuning role in platelet activation by regulating GPVI membrane dynamics.