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Supplementary Data from 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)

PDF file - 120K, C57Bl/6 mice bearing B16-Ova tumors were injected intratumorally with the respective viruses as described in materials and methods. Lymph nodes, spleens and tumors were collected 8 days after first virus injection, smashed and cultured for 24 hours. Analysis of T-cell markers was performed by flow cytometry for CD3, CD4 and CD8 and plotted as mean +SEM. *, P

PDF file - 91K, Nude mice bearing MB49 tumors were injected with Ad5/3-CMV-mCD40L, Ad5/3-Luc1 or PBS (Mock) on days 0,2 and 4. Eight days after first virus injection mice were killed and spleens and tumors were collected, smashed and cultured for 24 hours. Homogenates were analyzed for NK+ cells (left side) and CD19+ (right side). Results are presented as mean from 6 mice + SEM. *, P

PDF file - 2.2MB, (A) Cell monolayers were infected with 100 viral paticles /cell (VP/cell) and 24 hours later cells were analyzed by flow-cytometry measuring Annexin-V expression. Results are presented as percentage of positive cells relative to Mock uninfected cells. **, P

Supplementary Figure Legends 1-5, Table Legend from Suppression of Integrin α3β1 in Breast Cancer Cells Reduces Cyclooxygenase-2 Gene Expression and Inhibits Tumorigenesis, Invasion, and Cross-Talk to Endothelial Cells

Supplementary Figure 2 from Suppression of Integrin α3β1 in Breast Cancer Cells Reduces Cyclooxygenase-2 Gene Expression and Inhibits Tumorigenesis, Invasion, and Cross-Talk to Endothelial Cells

Supplementary Figure 3 from Suppression of Integrin α3β1 in Breast Cancer Cells Reduces Cyclooxygenase-2 Gene Expression and Inhibits Tumorigenesis, Invasion, and Cross-Talk to Endothelial Cells

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PDF file - 128K, T-cell analyses in C57Bl/6 mice bearing B16-Ova tumors C57Bl/6 mice bearing B16-Ova tumors were injected intratumorally. Lymph nodes, spleens and tumors were collected 8 days after first virus injection, homogenized and cultured for 24 hours. Analysis of Ova-specific CD8+CD19- was performed by flow cytometry gating the CD19- population and plotting SIINFEKEL marker of Ova with CD8. Results are represented as percentage of double positive CD8 and SIINFEKEL peptide and plotted as mean +SEM. *, P

Supplementary Table 1 from Suppression of Integrin α3β1 in Breast Cancer Cells Reduces Cyclooxygenase-2 Gene Expression and Inhibits Tumorigenesis, Invasion, and Cross-Talk to Endothelial Cells

Supplementary Figure 5 from Suppression of Integrin α3β1 in Breast Cancer Cells Reduces Cyclooxygenase-2 Gene Expression and Inhibits Tumorigenesis, Invasion, and Cross-Talk to Endothelial Cells

Supplementary Figure 1 from Suppression of Integrin α3β1 in Breast Cancer Cells Reduces Cyclooxygenase-2 Gene Expression and Inhibits Tumorigenesis, Invasion, and Cross-Talk to Endothelial Cells

PDF file - 75K, (A) MB49 cells were infected with 100 viral particles/cell with Ad5/3-CMV-mCD40L and Ad5/3-Luc1 and Annexin-V was assessed 24 hours post-infection by flow cytometry. (B) Replication of Ad5/3-CMV-mCD40L and Ad5/3-Luc1 over time in MB49 cell line. Cell monolayers were infected at doses specified ranging from 101 to 108 viral particles and assays were stopped at different time points (days 2, 3, 4 and 8). Cell viability was measured and expressed overtime. (C) MB49 cells were infected with 100 viral particles and samples were collected at 0, 24, 48 and 72 hours post infection. Quantitative PCR was performed for E4 sequence and values were normalized to mouse beta-actin gene.

Supplementary Figure 4 from Suppression of Integrin α3β1 in Breast Cancer Cells Reduces Cyclooxygenase-2 Gene Expression and Inhibits Tumorigenesis, Invasion, and Cross-Talk to Endothelial Cells

Integrin receptors for cell adhesion to extracellular matrix have important roles in promoting tumor growth and progression. Integrin α3β1 is highly expressed in breast cancer cells in which it is thought to promote invasion and metastasis; however, its roles in regulating malignant tumor cell behavior remain unclear. In the current study, we used short-hairpin RNA (shRNA) to show that suppression of α3β1 in a human breast cancer cell line, MDA-MB-231, leads to decreased tumorigenicity, reduced invasiveness, and decreased production of factors that stimulate endothelial cell migration. Real-time PCR revealed that suppression of α3β1 caused a dramatic reduction in expression of the cyclooxygenase-2 (COX-2) gene, which is frequently overexpressed in breast cancers and has been exploited as a therapeutic target. Decreased COX-2 was accompanied by reduced prostaglandin E2 (PGE2), a major prostanoid produced downstream of COX-2 and an important effector of COX-2 signaling. shRNA-mediated suppression of COX-2 showed that it has a role in tumor cell invasion and cross-talk to endothelial cells. Furthermore, treatment with PGE2 restored these functions in α3β1-deficient MDA-MB-231 cells. These findings identify a role for α3β1 in regulating two properties of tumor cells that facilitate cancer progression: invasiveness and ability to stimulate endothelial cells. They also reveal a novel role for COX-2 as a downstream effector of α3β1 in tumor cells, thereby identifying α3β1 as a potential therapeutic target to inhibit breast cancer. Cancer Res; 70(15); 6359–67. ©2010 AACR.

PDF file - 881K, (A) Oncolytic potency of Ad5/3-hTERT-E1A-hCD40L and (B) Ad5/3-hTERT-E1A on EJ (CD40+) and A549 (CD40-). Cell lines were infected with Ad5/3Luc1, Ad5/3-hTERT-E1A, Ad5/3-hTERT-E1A-hCD40L and Ad5/3-CMV-hCD40L and cell viability was measured by MTS assay The assay was stopped 7 days after infection and the data was plotted to compare efficacy on CD40+ versus CD40- cells; Ad5/3-hTERT-E1A-hCD40L was more effective than the positive control on the former while the opposite was true for the latter. . VP=viral particles. ***, P

The oncogenic pathways activated by the NPM-ALK chimeric kinase of ALK+ anaplastic large cell lymphoma (ALCL) are well characterized; however, the potential interactions of ALK signaling with the microenvironment are not yet known. Here we report that ALK+ ALCL-derived exosomes contain critical components of ALK signaling as well as CD30, and that exosome uptake by lymphoid cells led to increased proliferation and expression of critical antiapoptotic proteins by the recipient cells. The bone marrow fibroblasts highly uptake ALK+ ALCL-derived exosomes and acquire a cancer-associated fibroblast (CAF) phenotype. Moreover, exosome-mediated activation of stromal cells altered the cytokine profile of the microenvironment. These interactions may contribute to tumor aggressiveness and possibly resistance to treatment.

The oncogenic pathways activated by the NPM-ALK chimeric kinase of ALK+ anaplastic large cell lymphoma (ALCL) are well characterized; however, the potential interactions of ALK signaling with the microenvironment are not yet known. Here we report that ALK+ ALCL-derived exosomes contain critical components of ALK signaling as well as CD30, and that exosome uptake by lymphoid cells led to increased proliferation and expression of critical antiapoptotic proteins by the recipient cells. The bone marrow fibroblasts highly uptake ALK+ ALCL-derived exosomes and acquire a cancer-associated fibroblast (CAF) phenotype. Moreover, exosome-mediated activation of stromal cells altered the cytokine profile of the microenvironment. These interactions may contribute to tumor aggressiveness and possibly resistance to treatment.

Signaling through adhesion-related molecules is important for cancer growth and metastasis and cancer cells are resistant to anoikis, a form of cell death ensued by cell detachment from the extracellular matrix. Herein, we report that detached carcinoma cells and immortalized fibroblasts display defects in TNF and CD40 ligand (CD40L)-induced MEK-ERK signaling. Cell detachment results in reduced basal levels of the MEK kinase TPL2, compromises TPL2 activation and sensitizes carcinoma cells to death-inducing receptor ligands, mimicking the synthetic lethal interactions between TPL2 inactivation and TNF or CD40L stimulation. Focal Adhesion Kinase (FAK), which is activated in focal adhesions and mediates anchorage-dependent survival signaling, was found to sustain steady state TPL2 protein levels and to be required for TNF-induced TPL2 signal transduction. We show that when FAK levels are reduced, as seen in certain types of malignancy or malignant cell populations, the formation of cIAP2:RIPK1 complexes increases, leading to reduced TPL2 expression levels by a dual mechanism: first, by the reduction in the levels of NF-κΒ1 which is required for TPL2 stability; second, by the engagement of an RelA NF-κΒ pathway that elevates interleukin-6 production, leading to activation of STAT3 and its transcriptional target SKP2 which functions as a TPL2 E3 ubiquitin ligase. These data underscore a new mode of regulation of TNF family signal transduction on the TPL2-MEK-ERK branch by adhesion-related molecules that may have important ramifications for cancer therapy.