Abstract PR03: Targeting multiple cell cycle regulatory points for the prevention of triple-negative breast cancer
Authors: Litzenburger Beate Christiane , Uray Ivan Peter , Hill Jamal , Zhang Jun , Mazumdar Abhijit , Brown Powel H. 
Published: October 2015

Abstract
Background: Anti-estrogen drugs such as selective estrogen receptor modulators and aromatase inhibitors effectively prevent ER-positive breast cancer, but do not prevent triple negative breast cancer (TNBC) which represents approximately 20% of all breast cancers. TNBCs are characterized by a high proliferation rate and a dysregulated cell cycle. Drugs that specifically activate RXR without activating RAR nuclear receptors (“rexinoids”) are being studied as potential cancer preventive drugs. One of the most promising rexinoids is the compound, LG100268. LG100268 is highly effective in preventing ER-negative/HER2-positive mammary tumors in MMTV-ErbB2 mice by inhibiting cyclin D-dependent pathways, but less preventive in models of TNBC (p53-null mice and C3(1)-SV40 Tag mice). Therefore, we hypothesized that inhibiting multiple cell cycle regulatory points by combining kinase inhibitors and LG100268 is an effective and safe strategy to prevent triple negative breast cancer.
Methods: To identify kinases that inhibit cell growth we carried out a ‘whole kinome’ (636 kinases) high-throughput siRNA screen in combination with LG100268 (1µM) in immortalized, human mammary epithelial cells overexpressing telomerase (HME-tert). After 4 days of growth following LG100268 treatment and suppression of the respective kinase cells were stained with DAPI and cell number was determined by automated microscopy and image analysis. Combination “hits” were selected based on a p-value &lt;0.05, FDR &lt;0.05 compared to siRNA treatment alone. Transfections with siRNAs to kinase genes were repeated to confirm gene silencing using quantitative real-time PCR (qRT-PCR) and analyzed for cell growth in combination with 1µM LG100268 1-4 days post transfection. These studies identified cyclin dependent kinases as potential target that when inhibited showed additivity with LG100268. In vivo studies were performed using C3(1)-SV40 Tag transgenic mice. 10 week old female mice were treated i.p. 3x/week with a CDK inhibitor dinaciclib (20 mg/kg) or vehicle, and 5x/week with LG100268 (25mg/kg) or vehicle. Mice were observed for tumor incidence and toxicity and euthanized 2 months after treatment, or if tumor volume reached 1500mm3. Mammary glands were analyzed for the incidence of early lesions using Image J. Tumors were detected as a palpable mammary mass with a volume of at least 100 mm3. Survival analyses used log-rank tests, and Kaplan–Meier curves were plotted using GraphPad.
Results: The analysis of the kinome screen showed that the combination of LG100268 with each of 27 kinases had a significant growth suppressive effect compared to siRNA control and vehicle alone. Knockdown of the respective kinases was independently confirmed by qRT PCR. Growth assays of HME-tert cells after siRNA knockdown of kinases in combination with LG100268 demonstrated that in particular CDK1, 2 and 5 enhanced the chemopreventive effect of LG100268. Using a CDK inhibitor dinaciclib in combination with LG100268 showed that the combination additively suppressed mammary epithelial growth in vitro. In vivo in SV40 Tag mice, LG100268 or dinaciclib alone significantly reduced the area (mm2) of hyperplastic lesions (defined as any hyperplastic ductal foci comprised of more than three layers of epithelial cells) compared to the control group. The combination therapy did not result in a significant reduction of hyperplastic area compared to dinaciclib alone. Long-term treatment with LG100268 did not delay TNBC formation compared to vehicle (both MTTF = 144 days). However, long-term treatment with dinaciclib alone delayed mammary tumorigenesis with a median time to tumor formation (MTTF) of 167 days compared to a MTTF of 144 days of age in the control group (p &lt; 0.001). The combination therapy did not further affect the tumor formation compared to dinaciclib alone (MTTF = 162 days).
Conclusions: These studies demonstrate that dinaciclib alone or the combination therapy of LG100268 and dinaciclib causes inhibition of premalignant cell growth and delayed ER-negative mammary tumorigenesis in SV40 Tag mice. While an additive effect of dinaciclib and LG100268 was observed in vitro, the in vivo combination therapy was not significantly more effective than dinaciclib treatment alone. Future studies should investigate different combinations of dosages of dinaciclib and LG100268 in vivo. These studies were supported by an NCI/NIH R25T grant (R25CA057730, BL) and Susan G. Komen for the Cure Promise grant (KG081694)
Citation Format: Beate Christiane Litzenburger, Ivan Peter Uray, Jamal Hill, Jun Zhang, Abhijit Mazumdar, Powel H. Brown. Targeting multiple cell cycle regulatory points for the prevention of triple-negative breast cancer. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr PR03.