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. 2008 May 1;68(9):3243-50.
doi: 10.1158/0008-5472.CAN-07-5480.

Cancer stem cells contribute to cisplatin resistance in Brca1/p53-mediated mouse mammary tumors

Norazizah Shafee  1 , Christopher R Smith, Shuanzeng Wei, Yoon Kim, Gordon B Mills, Gabriel N Hortobagyi, Eric J Stanbridge, Eva Y-H P Lee
Affiliations

Cancer stem cells contribute to cisplatin resistance in Brca1/p53-mediated mouse mammary tumors

Norazizah Shafee et al. Cancer Res. .

Abstract

The majority of BRCA1-associated breast cancers are basal cell-like, which is associated with a poor outcome. Using a spontaneous mouse mammary tumor model, we show that platinum compounds, which generate DNA breaks during the repair process, are more effective than doxorubicin in Brca1/p53-mutated tumors. At 0.5 mg/kg of daily cisplatin treatment, 80% primary tumors (n = 8) show complete pathologic response. At greater dosages, 100% show complete response (n = 19). However, after 2 to 3 months of complete remission following platinum treatment, tumors relapse and become refractory to successive rounds of treatment. Approximately 3.8% to 8.0% (mean, 5.9%) of tumor cells express the normal mammary stem cell markers, CD29(hi)24(med), and these cells are tumorigenic, whereas CD29(med)24(-/lo) and CD29(med)24(hi) cells have diminished tumorigenicity or are nontumorigenic, respectively. In partially platinum-responsive primary transplants, 6.6% to 11.0% (mean, 8.8%) tumor cells are CD29(hi)24(med); these populations significantly increase to 16.5% to 29.2% (mean, 22.8%; P < 0.05) in platinum-refractory secondary tumor transplants. Further, refractory tumor cells have greater colony-forming ability than the primary transplant-derived cells in the presence of cisplatin. Expression of a normal stem cell marker, Nanog, is decreased in the CD29(hi)24(med) populations in the secondary transplants. Top2A expression is also down-regulated in secondary drug-resistant tumor populations and, in one case, was accompanied by genomic deletion of Top2A. These studies identify distinct cancer cell populations for therapeutic targeting in breast cancer and implicate clonal evolution and expansion of cancer stem-like cells as a potential cause of chemoresistance.

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Figures

Figure 1
Figure 1
Brca1/p53–mediated mammary tumors are sensitive to cisplatin treatment. A, proportion of tumor-free nulliparous Brca1fp/fpp53fp/fp and p53fp/fp;Cre mice. X axis, age of each mouse when a palpable tumor was first detected. The curves were plotted using the Kaplan-Meier method. n values indicate the number of mice analyzed of each genotype. B, response of p53-mediated (top) and Brca1/p53–mediated (bottom) mammary tumors to doxorubicin or CDDP in vivo. Data were obtained using both WAPCrec and MMTVCrea mice. Tumor growth was monitored by taking daily caliper measurements in two perpendicular dimensions as described in Materials and Methods. C, immunohistochemical staining of activated caspase-3 (Casp-3) and phospho-histone 3 (P-H3) in CDDP- or doxorubicin (DOX)-treated samples at the indicated time points. D, Rad51 foci formation in CDDP- or doxorubicin-treated tumors. Insets, higher magnifications of stained nuclei. A total of 8 Brca1/p53–mediated and 10 p53-mediated tumors were analyzed, and all showed similar results.
Figure 2
Figure 2
Chemoresistance to CDDP in vivo and the presence of cancer stem cells. A, sensitivity of primary and recurrent mammary tumors to CDDP treatment. CDDP was administered i.p., when tumors reached ~0.5 cm3, at 0.5 mg/kg daily for 7 d. Tumor sizes were monitored following detection of palpable tumors before, during, and after CDDP treatment (n = 8). Tumor that recurred in three mice underwent additional cycle of treatments. B, FACS sorting analyses done on cells dissociated from primary tumor. Cells sorted according to the indicated gatings were used for in vivo tumorigenicity assays. C, transplantation using 1,000 cells of the CD29hiCD24med subpopulation resulted in tumor formation (arrows) whereas CD29med24–/lo (arrowhead) or CD29med24hi (asterisk) did not. D, a representative FACS profile of the resulting tumors following CD29hiCD24med cell transplantations.
Figure 3
Figure 3
Chemoresponse, FACS profiling, and colony-forming abilities of the primary and secondary Brca1fp/fpp53fp/fp tumor transplants. A, response to CDDP treatment of the primary and secondary tumor transplants, developed after transplantation with 1,000 CD29hiCD24med cells. The treatment was administered i.p., when tumors reached ~0.5 cm3,at 0.5 mg/kg daily for 7 d. Tumor volumes before, during, and after CDDP treatment were shown. Arrows are not drawn to scale. B, FACS analyses of cells dissociated from primary tumor transplant, which showed partial resistance to CDDP (left), and from secondary tumor transplant, which were refractory to CDDP (right). C, colony formation assay of sorted CD29hiCD24med, CD29med24–/lo, and CD29med24hi cells from the primary tumor transplant. D, colony formation of unsorted cells from primary and secondary tumor transplants in the presence or absence of CDDP. C and D, columns, mean number of colonies per 100 primary cells; bars, SD. *, P < 0.05, versus the control group.
Figure 4
Figure 4
Selective gene expression profiles in recurrent primary and secondary tumor transplants. A, semiquantitative RT-PCR analyses of CD29hiCD24med, CD29med24–/lo, and CD29med24hi cells from the primary and secondary tumor transplants. B, a positive control for Oct4, using mouse embryonic stem (ES) cells. C, semiquantitative PCR analyses of genomic DNA of CD29hiCD24med cells from the primary and secondary tumor transplants; WT, wild-type. D, model illustrating changes in cancer stem cells during the course of chemotherapy.
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