Alpha5beta1 Integrin and Oncofetal Fibronectin are Upregulated in Malignant Breast Cells in...
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Results: Gene array analysis comparing 6D12+ vs. 6D12- populations revealed significant changes in 600 genes. Gene array analysis comparing spheres vs. matched non-tumorigenic in vitro epithelial cells revealed significant changes in 3100 genes. The tumorigenic 6D12- and sphere populations shared 160 gene changes, against a background of 1-4 shared gene changes for non-congruent groups. To rank these genes for clinical and biological significance, we curated publicly available gene array da- tabases for advanced EOC with clinical outcome correlates. Parametric p values based on single gene Cox hazard ratios were gen- erated. The resultant genes were then filtered for surface expression to produce a list of candidate markers of the CSC population, to be confirmed by single cell expression profile and xenograft modeling. Conclusions: Merging traditional purification methods with gene array analysis, and publicly available outcomes data may speed discovery of CSC markers. Identification of surface markers permits generation of imaging reagents for target identification, as well as radioimmunological reagents for direct therapy. Identification of the CSC population itself permits identification and inhibition of active radioresistance pathways. Author Disclosure: R.K. Chin, None; D. Sahoo, None; S. Willingham, None; T. Storm, None; S. Schmidt, None; J. Ross, None; K. Yang, None; L. Ailles, None; N. Teng, None; I. Weissman, None. 2807 Alpha5beta1 Integrin and Oncofetal Fibronectin are Upregulated in Malignant Breast Cells in Three- dimensional Culture That can be Specifically Targeted for Radiation Therapy J. Nam 1 C. C. Park 2 1 Lawrence Berkeley National Laboratory, Berkeley, CA, 2 University of California, San Francisco, San Francisco, CA Purpose/Objective(s): Integrin beta1 signaling has been implicated in breast cancer progression and has been shown to facilitate resistance to radiation that could be abrogated using beta1 integrin inhibitory antibody, AIIB2. In the present study, we investigated whether a specific alpha heterodimer of beta1 integrin was preferentially mediating a pro-survival signal in human breast cancer cells, and if it could specifically be targeted for radiation therapy. Materials/Methods: We used a three-dimensional laminin rich extracellular matrix (3D lrECM) culture to compare the relative levels of alpha heterodimer expression in malignant HMT3522-T4-2 and nonmalignant cell line HMT3522-S-1. Total cell lysates were probed by western blot and subjected to immunoprecipitation to detect several possible alpha subunits. In addition, to inves- tigate whether alpha5beta1 integrin mediated an important pro-survival signal, we treated 3D lrECM cultures with a small peptide inhibitor of alpha5beta1 integrin and fibronectin (FN) interaction, Ac-PHSCN-NH2 (ATN-161) and measured apoptotic response using TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated nick end labeling) assay. Results: We found that expression of alpha5beta1 integrin and its ligand, FN were upregulated in T4-2 malignant breast cell line compared to nonmalignant cell line S1. We further discovered that oncofetal FN, containing the EDA+ domain was specifically over-expressed in malignant T4-2 cells in 3D lrECM culture. Apoptosis was increased in T4-2 cells cultured in 3D lrECM by ATN- 161 treatment at 0.5 mg/ml (mean=8.3%, p \0.001) or 2.0 mg/ml (mean=23.4%, p \0.001). Moreover, treatment with 0.5 mg/ml ATN-161 and 2 Gy radiation together increase apoptosis compared to single treatment in breast cancer cell lines, T4-2 cells, and MDA-MB-231 cells. Conclusions: We have previously shown that inhibitory antibody to beta1 integrin, AIIB2, leads to selective apoptosis and de- creased proliferation in human breast cancer cells in 3DlrECM culture and in vivo. In addition, combining beta1 integrin inhibition with IR allowed for reduction of IR dose necessary to achieve growth inhibition in vivo. Here, we show that in HMT3522-T4-2 breast cancer cell line, inhibition of alpha5beta1 integrin and FN interaction using ATN-161 significantly enhanced apoptosis in 3D lrECM. These data indicate that FN and its receptor, alpha5beta1 integrin play a pivotal role for survival signaling in breast cancer and may be an important target to enhance radiotherapy efficacy. Author Disclosure: J. Nam, None; C.C. Park, None. 2808 Identification of Conserved Gene Expression Programs in Epithelial Cancer Stem Cells M. Diehn 1 , R. W. Cho 1 , L. Ailles 2 , J. S. Lam 1 , M. J. Kaplan 1 , G. Somlo 3 , I. L. Weissman 1 , M. F. Clarke 1 1 Stanford University School of Medicine, Stanford, CA, 2 Ontario Cancer Institute, Toronto, ON, Canada, 3 Hope National Medical Center, Duarte, CA Purpose/Objective(s): The cancer stem cell (CSC) hypothesis proposes that tumors are maintained by a subset of cancer cells which has the ability to proliferate indefinitely and which can give rise to non-tumorigenic cancer cells (NTCs) with limited pro- liferative potential. Although normal stem cells are not necessarily the target cell of transformation, a growing body of evidence indicates that there are significant similarities between normal stem cells and CSCs. We hypothesized that CSCs from different tumor types display conserved gene expression programs and that these are related to normal stem cell gene expression profiles. Materials/Methods: We performed gene expression profiling of human CSCs from primary breast and head & neck cancers to explore relationships between CSCs and normal stem cells. We employed unsupervised and supervised analyses to evaluate the data. Specifically, we searched for conserved CSC gene expression patterns between the two tumor types and compared CSC gene expression profiles to those of normal stem cells. Finally, we used clonogenic survival assays to assess CSC sensitivity to candidate self-renewal/survival pathway inhibition. Results: Unsupervised analyses of gene expression profiles of primary human breast and head & neck CSCs revealed that CSCs from the two types of cancers were more similar to each other than to their corresponding NTCs. Supervised analyses confirmed the existence of conserved gene expression programs in CSCs from different types of carcinomas. The CSC gene expression signature was strongly enriched for genes expressed by normal adult tissue stem cells, including mouse mammary stem cells and human hematopoietic stem cells. Furthermore, genes implicated in stem cell self-renewal were highly overrepresented in the CSC signa- ture. Inhibition of self-renewal pathways significantly reduced clonogenicity of breast CSCs. Conclusions: Our findings indicate that CSCs from different types of carcinomas express a conserved gene expression program which includes genes involved in stem cell self-renewal. Targeting of self-renewal-associated pathways in CSCs represents S544 I. J. Radiation Oncology d Biology d Physics Volume 75, Number 3, Supplement, 2009
Transcript of Alpha5beta1 Integrin and Oncofetal Fibronectin are Upregulated in Malignant Breast Cells in...
S544 I. J. Radiation Oncology d Biology d Physics Volume 75, Number 3, Supplement, 2009
Results: Gene array analysis comparing 6D12+ vs. 6D12- populations revealed significant changes in �600 genes. Gene arrayanalysis comparing spheres vs. matched non-tumorigenic in vitro epithelial cells revealed significant changes in �3100 genes.The tumorigenic 6D12- and sphere populations shared 160 gene changes, against a background of 1-4 shared gene changes fornon-congruent groups. To rank these genes for clinical and biological significance, we curated publicly available gene array da-tabases for advanced EOC with clinical outcome correlates. Parametric p values based on single gene Cox hazard ratios were gen-erated. The resultant genes were then filtered for surface expression to produce a list of candidate markers of the CSC population, tobe confirmed by single cell expression profile and xenograft modeling.
Conclusions: Merging traditional purification methods with gene array analysis, and publicly available outcomes data may speeddiscovery of CSC markers. Identification of surface markers permits generation of imaging reagents for target identification, as wellas radioimmunological reagents for direct therapy. Identification of the CSC population itself permits identification and inhibitionof active radioresistance pathways.
Author Disclosure: R.K. Chin, None; D. Sahoo, None; S. Willingham, None; T. Storm, None; S. Schmidt, None; J. Ross, None; K.Yang, None; L. Ailles, None; N. Teng, None; I. Weissman, None.
2807 Alpha5beta1 Integrin and Oncofetal Fibronectin are Upregulated in Malignant Breast Cells in Three-
dimensional Culture That can be Specifically Targeted for Radiation TherapyJ. Nam1 C. C. Park2
1Lawrence Berkeley National Laboratory, Berkeley, CA, 2University of California, San Francisco, San Francisco, CA
Purpose/Objective(s): Integrin beta1 signaling has been implicated in breast cancer progression and has been shown to facilitateresistance to radiation that could be abrogated using beta1 integrin inhibitory antibody, AIIB2. In the present study, we investigatedwhether a specific alpha heterodimer of beta1 integrin was preferentially mediating a pro-survival signal in human breast cancercells, and if it could specifically be targeted for radiation therapy.
Materials/Methods: We used a three-dimensional laminin rich extracellular matrix (3D lrECM) culture to compare the relativelevels of alpha heterodimer expression in malignant HMT3522-T4-2 and nonmalignant cell line HMT3522-S-1. Total cell lysateswere probed by western blot and subjected to immunoprecipitation to detect several possible alpha subunits. In addition, to inves-tigate whether alpha5beta1 integrin mediated an important pro-survival signal, we treated 3D lrECM cultures with a small peptideinhibitor of alpha5beta1 integrin and fibronectin (FN) interaction, Ac-PHSCN-NH2 (ATN-161) and measured apoptotic responseusing TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated nick end labeling) assay.
Results: We found that expression of alpha5beta1 integrin and its ligand, FN were upregulated in T4-2 malignant breast cell linecompared to nonmalignant cell line S1. We further discovered that oncofetal FN, containing the EDA+ domain was specificallyover-expressed in malignant T4-2 cells in 3D lrECM culture. Apoptosis was increased in T4-2 cells cultured in 3D lrECM by ATN-161 treatment at 0.5 mg/ml (mean=8.3%, p\0.001) or 2.0 mg/ml (mean=23.4%, p\0.001). Moreover, treatment with 0.5 mg/mlATN-161 and 2 Gy radiation together increase apoptosis compared to single treatment in breast cancer cell lines, T4-2 cells, andMDA-MB-231 cells.
Conclusions: We have previously shown that inhibitory antibody to beta1 integrin, AIIB2, leads to selective apoptosis and de-creased proliferation in human breast cancer cells in 3DlrECM culture and in vivo. In addition, combining beta1 integrin inhibitionwith IR allowed for reduction of IR dose necessary to achieve growth inhibition in vivo. Here, we show that in HMT3522-T4-2breast cancer cell line, inhibition of alpha5beta1 integrin and FN interaction using ATN-161 significantly enhanced apoptosis in 3DlrECM. These data indicate that FN and its receptor, alpha5beta1 integrin play a pivotal role for survival signaling in breast cancerand may be an important target to enhance radiotherapy efficacy.
Author Disclosure: J. Nam, None; C.C. Park, None.
2808 Identification of Conserved Gene Expression Programs in Epithelial Cancer Stem Cells
M. Diehn1, R. W. Cho1, L. Ailles2, J. S. Lam1, M. J. Kaplan1, G. Somlo3, I. L. Weissman1, M. F. Clarke1
1Stanford University School of Medicine, Stanford, CA, 2Ontario Cancer Institute, Toronto, ON, Canada, 3Hope NationalMedical Center, Duarte, CA
Purpose/Objective(s): The cancer stem cell (CSC) hypothesis proposes that tumors are maintained by a subset of cancer cellswhich has the ability to proliferate indefinitely and which can give rise to non-tumorigenic cancer cells (NTCs) with limited pro-liferative potential. Although normal stem cells are not necessarily the target cell of transformation, a growing body of evidenceindicates that there are significant similarities between normal stem cells and CSCs. We hypothesized that CSCs from differenttumor types display conserved gene expression programs and that these are related to normal stem cell gene expression profiles.
Materials/Methods: We performed gene expression profiling of human CSCs from primary breast and head & neck cancers toexplore relationships between CSCs and normal stem cells. We employed unsupervised and supervised analyses to evaluate thedata. Specifically, we searched for conserved CSC gene expression patterns between the two tumor types and compared CSCgene expression profiles to those of normal stem cells. Finally, we used clonogenic survival assays to assess CSC sensitivity tocandidate self-renewal/survival pathway inhibition.
Results: Unsupervised analyses of gene expression profiles of primary human breast and head & neck CSCs revealed that CSCsfrom the two types of cancers were more similar to each other than to their corresponding NTCs. Supervised analyses confirmed theexistence of conserved gene expression programs in CSCs from different types of carcinomas. The CSC gene expression signaturewas strongly enriched for genes expressed by normal adult tissue stem cells, including mouse mammary stem cells and humanhematopoietic stem cells. Furthermore, genes implicated in stem cell self-renewal were highly overrepresented in the CSC signa-ture. Inhibition of self-renewal pathways significantly reduced clonogenicity of breast CSCs.
Conclusions: Our findings indicate that CSCs from different types of carcinomas express a conserved gene expression programwhich includes genes involved in stem cell self-renewal. Targeting of self-renewal-associated pathways in CSCs represents
