Abstract B011: IGFR as a candidate therapeutic target for cancer-initiating cells in triple-negative breast cancer
Authors: Ueno Naoto T. , Kai Kazuharu 
Published: May 2015

Background: Cancer-initiating cells (CICs) play a crucial role in recurrence of triple-negative breast cancer (TNBC). Thus, therapies targeting CICs may help prevent TNBC recurrence. However, the molecular characteristics of TNBC CICs have not been fully elucidated. We sought to determine molecular traits of TNBC CICs by establishing a mouse model of TNBC that successfully recapitulates human TNBC.

Methods: In TNBC, Rb and p53 pathways are frequently abrogated, and intracellular kinases such as Akt and MAPK are frequently activated. To recapitulate these genetic components, we used Ink4a/Arf-knockout (KO) mouse mammary epithelial cells (MECs). Ink4a/Arf transcribes p16Ink4a and p19Arf and activates Rb and p53; therefore, Ink4a/Arf-KO mimics abrogation of Rb and p53 pathways. In addition, we retrovirally introduced oncogenic HRAS(G12V), which is a robust activator of cytoplasmic kinases, including Akt and MAPK, into Ink4a/Arf-KO MECs in vitro. Mammospheres of HRAS transfected Ink4a/Arf-KO MECs were studied for expression patterns CD29, CD24, CD44, Sca1, CD61, and CD49f in tumor cells by FACS. Tumor cells were sorted according to their protein expression levels and inoculated into mammary fat pads of recipient mice to determine tumor-initiating activities of each subpopulation. The molecular profiles of CICs were determined by mouse microarray expression analysis.

Results: Unexpectedly, CD49f-negative (CD49f-) tumor cells had much higher tumor-initiating activity than CD49f-positive (CD49f+) cells at 100-cell inoculations (CD49f-, 5/8 mice; CD49f+, 0/8 mice). We hypothesized that tumor initiation from CD49f- cells depends on their quiescent profile, which is a trait of CICs. To test this hypothesis, we further fractionated CD49f- cells into quiescent (CD49f-q) cells and dividing cells and inoculated these fractions into recipient mice. CD49f-q cells had higher tumor-initiating activity than CD49f- dividing cells. We concluded that CD49f-q cells were CICs in our model. Genomewide expressions of CD49f-q cells and CD49f+ cells were compared by paired t-test, and over- and underexpressed genes in CD49f-q cells were functionally annotated using ingenuity pathway analysis. One of the top-ranked bioprocesses of overexpressed genes in CD49f-q cells was the IGF-1 pathway. On the basis of these findings, we tested an IGF-1R/InsR dual kinase inhibitor in TNBC. We confirmed high IGF-1R and low InsR expression in TNBC cell lines (e.g., SUM149, MDA-MB-231, MDA-MB-468). With the IGF-1R/InsR dual kinase inhibitor, proportions of CICs, profiled as CD44+CD24-, were significantly reduced in a dose-dependent manner in SUM149 and MDA-MB-231 cells. Indeed, the dual kinase inhibitor dose-dependently inhibited in vitro mammosphere formation, a hallmark of CIC activity. An in vitro growth inhibition assay revealed that IGF-1R/InsR dual kinase inhibitor inhibited cell growth (all IC50 &lt;0.5 μM), and an agar assay for colony formation confirmed this antitumor effect in TNBC cells.

Conclusion: We found that CICs in TNBC function in part through the IGF-1 pathway. We found that an IGF-1R/InsR dual kinase inhibitor had significant antitumor effects via suppressing the human CIC phenotype.

Citation Format: Naoto T. Ueno, Kazuharu Kai. IGFR as a candidate therapeutic target for cancer-initiating cells in triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B011.