(233an) Evaluation of the Therapeutic Potential of FOXC1 Knockdown for Treatment of Basal-Like Breast Cancers

Authors: 
Mott, L. A., University of Kentucky
Basal-like breast cancer (BLBC) accounts for 15% of breast cancer diagnoses, is characterized as one of the most aggressive forms of breast cancer, and exhibits the worst prognosis. The cancer tends to metastasize to the brain and lungs during early stages [1]. Due to the BLBC triple negative phenotype (estrogen receptor-, progesterone receptor-, human epidermal growth factor 2-), chemotherapy remains the only treatment despite its poor success rate and propensity for relapse. The aggressive growth and mobility of BLBC contribute to its difficulty of treatment and result in low survivability [2-3]. This mortality indicates the urgency for new therapeutic approaches to replace or supplement the limited chemotherapeutic treatment options. Since the forkhead-box transcription factor C1 (FOXC1) was isolated as a unique biomarker for BLBC [4], alternative therapeutic approaches focusing on inhibition of FOXC1 have emerged. FOXC1 overexpression in BLBC was linked to excessive cell proliferation and aggressive mobility. Knockdown of FOXC1 using RNA interference, in contrast, was shown to decrease proliferation and mobility of BLBC cell lines in vitro. This suggests that the elimination or knockdown of FOXC1 is a potential therapeutic approach to the treatment of BLBC. This project evaluates this therapeutic potential both in vitro and in vivo by various gene therapy methods.

Knockdown of protein products from aberrant or overexpressed genes can be achieved by delivering repair machinery to act at the DNA level, or RNA interference machinery to elicit degradation of mRNA products of targeted genes. The CRISPR/Cas9 system is a cutting-edge method that has the ability to produce double stranded cuts in DNA at targeted locations in the eukaryotic genome in order to produce controlled deletions or insertions of genetic code. RNAi was used to knockdown FOXC1 in 4T1 cells (mouse BLBC). Knockdown (>95%) was verified via western blot and proliferation studies showed a statistically significant (p<0.013) reduction in cell growth up to 96 hours post transfection. The CRISPR/Cas9 system was used to produce clone 4T1 cells exhibiting knockout of the FOXC1 gene (verified via western blot). Clone proliferation studies in vitro showed similar results to knockdown studies. These clones were used to produce tumors in mice models that were compared to 4T1 tumors in control specimens. Knockout tumors exhibited no reduction in metastatic activity compared to controls and showed no statistical reduction in growth rate. The study indicates that therapies targeting FOXC1 are possible and even successful in vitro. However, the inherent complexity of physiological systems convolutes the effect of any positive therapeutic response in vivo.

1. Smid M, et al. Cancer Res. 2008; 68:3108-3114

2. Seewaldt VL, Scott V. N Engl J Med 2007; 356: e12

3. Sarrio D, et al. Cancer Res 2008; 68:989-997.

4. Ray P, et al. Cancer Res 2010; 70:3870-3876