Stem Cells of Molecularly Diverse ER-Negative Breast Cancers

Institution: Stanford University
Investigator(s): Stefanie Jeffrey, M.D. -
Award Cycle: 2005 (Cycle 11) Grant #: 11IB-0175 Award: $234,165
Award Type: IDEA
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2005)
The concept that breast cancer develops from cancer stem cells (CSCs) is gaining wider acceptance. Although CSCs comprise only a small fraction of a tumor, they have the capacity to self-renew and produce progeny that can expand or repopulate tumor bulk. In a mouse xenograft model, CSCs are tumorigenic, with as few as 100 CSCs able to reproduce a breast tumor similar to the original. In contrast, the remainder of malignant epithelial cells in a tumor mass are non-tumorigenic, and implanting 20,000 cells will not generate a breast tumor in the mouse model. Therefore, any therapy that kills cancer cells must eliminate the CSCs. Therapy that only kills CSC progeny, but not the CSCs, will shrink the tumor but not eradicate the cancer. It is unknown whether there are distinct types of CSCs that correspond to different molecular subtypes of breast cancer.

We hypothesize that molecularly diverse subtypes of breast cancer, as defined by DNA microarray expression profiling, are derived from molecularly distinct breast cancer stem cells. To address the question, we will study two molecularly diverse and clinically aggressive hormone receptor negative subtypes, basal-like and ERBB2-overexpressing tumors. We will ask whether the cancer stem cell populations from these two very different classes of breast cancers derive from a single estrogen receptor-negative (ER-) cancer stem cell or from different cancer stem cell populations. We will obtain tumor tissue from approximately 30 patients with confirmed hormone receptor negative (ER-/PR-) breast cancers and use DNA microarrays to identify the molecular subtypes of these tumors. CSCs from each of the tumor subtypes will be amplified in a mouse xenograft model and CSCs and non-CSC populations will be separated by fluorescent activated cell sorting (FACS). Limited-dilution assay will be used to confirm the tumorigenicity of CSCs and non-CSCs. The CSC and non-tumorigenic populations will be characterized by microarray analysis, immunohistochemisty, RNA in situ hybridization, and fluorescence in situ hybridization (FISH). We will use new computational methods to analyze and compare the genetic profiles of the different CSC and non-CSC populations and primary tumor for the basal-like and ERBB2-overexpressing tumor classes.

The characterization of cancer stem cells from different molecular subtypes of breast cancer has not been accomplished. It is imperative for the development of effective anti-cancer therapy, since the CSCs must be entirely eradicated if a breast cancer is to be cured. Genetic characterization of CSCs will pave the road towards targeted patient-specific treatment and may identify new avenues for chemoprevention, especially for ER- breast cancer, where there is currently no effective strategy.

Final Report (2008)
The overall goal of this project was to determine whether the cancer stem cell (CSC) populations for two distinct estrogen receptor (ER) negative breast cancer subtypes are different, and to describe the characteristics of the CSCs for each type. The subtypes to be studied were ER negative/HER2 negative and ER negative/HER2 positive. During the project period, a total of 24 breast cancer samples were obtained, including two samples from metastatic pleural effusions or ascites.

Fifteen human breast cancer specimens, including two specimens of cells from metastatic sites, were of sufficient quantity for implantation or injection into the mammary fat pads of NOD/SCID mice (orthotopic xenografts). Again, implantation of tiny tumor chunks into mice requires fresh tissue directly from the operating room and was performed at time of fresh tissue harvest without knowing hormone receptor status. Thus, nine tumors implanted into mice were hormone receptor negative and six were hormone receptor positive.

Four (67%) of the six “triple negative” solid tumor primary breast cancers generated xenograft tumors in mice. All four xenograft tumors plus the xenograft from the pleural effusion could be passaged by serial transplantation into other mice for multiple generations. Only one ER negative/HER2 positive tumor sample was accrued, and this tumor did not generate a xenograft, precluding additional studies on their cancer stem cells. Interestingly, of the six hormone receptor positive tumors implanted into mice, none formed xenografts. These results are consistent with (and for triple negative breast cancer, better than) recently published data from a French group who generated subcutaneous xenografts in athymic mice.

In most of our xenograft models, the histology of the primary tumors was fairly well conserved. Specifically, we compared cancer cell morphology, number of mitotic cells, stromal abundance, necrotic areas, and other features.

We then focused our research to ask the question whether cancer stem cells the same as circulating tumor cells. To accomplish this, we had to develop new technology to isolate, purify, and characterize circulating tumor cells. Circulating tumor cells (CTCs) are rare: less than 1 CTC per ml of blood. We have also captured and characterized CTCs from the blood of the NOD/SCID mice implanted with the fresh human breast cancers xenografts (generated as described above) as well as from the blood of patients with metastatic breast cancer. Our data indicate that single CTC gene expression may vary in a single human blood sample, possibly reflecting the heterogeneity of cells from single or multiple metastatic lesions. The expression profile of CTCs isolated from patient blood were compared to that of the primary tumor and, when available, to biopsies of metastases. In some patients, CTC profiles were similar to biomarkers in the primary tumor. However, in other cases the CTC profiles did not match the primary tumor profile, but instead reflected the profile of the biopsied metastasis.

Discovery and validation of breast cancer subtypes.
Periodical:BMC Genomics
Index Medicus: BMC Genomics
Authors: Kapp AV, Jeffrey SS, et al.
Yr: 2006 Vol: 7 Nbr: Abs: Pg:231

TP53 mutation status and gene expression profiles are powerful prognostic markers of breast cancer.
Periodical:Breast Cancer Research
Index Medicus: Breast Cancer Res
Authors: Langerød A, Zhao H, et al Jeffrey SS
Yr: 2007 Vol: 9 Nbr: 3 Abs: Pg:R30

Disease-specific genomic analysis: identifying the signature of pathologic biology.
Index Medicus:
Authors: Nicolau M, Tibshirani R, Børresen-Dale AL, Jeffrey SS
Yr: 2007 Vol: 23 Nbr: 8 Abs: Pg:957-65

Estrogen receptor-negative invasive breast cancer: imaging features of tumors with and without human epidermal growth factor receptor type 2 overexpression.
Index Medicus: Radiology
Authors: Wang Y, Ikeda DM, Narasimhan B, Longacre TA, Bleicher RJ, Pal S, Jackman RJ, Jeffrey SS
Yr: 2008 Vol: 246 Nbr: 2 Abs: Pg:367-75

RNA extraction from ten year old formalin-fixed paraffin-embedded breast cancer samples: a comparison of column purification and magnetic bead-based technologies.
Periodical:BMC Molecular Biology
Index Medicus: BMC Mol Biol
Authors: Ribeiro-Silva A, Zhang H, Jeffrey SS.
Yr: 2007 Vol: 8 Nbr: Abs: Pg:118

Oxidative stress pathways highlighted in tumor cell immortalization: association with breast cancer outcome.
Index Medicus: Oncogene
Authors: Dairkee SH, Nicolau M, Sayeed A, Champion S, Ji Y, Moore DH, Yong B, Meng Z, Jeffrey SS.
Yr: 2007 Vol: 26 Nbr: 43 Abs: Pg:6269-79