Background: Breast cancer is the leading worldwide cause of death among women due to the high metastatic spread of this disease. As by definition, Cancer Initiating Cells (CICs) are a fraction of primary tumor cells harboring tumorigenic potential and successful outgrowth at metastatic sites. Targeting molecular events affecting CICs peculiarities, as self-renewal and an innate chemoresistance, could improve the ineffectiveness of current therapies.

Sam68, the major CD44 splicing regulator, is a multifunctional RNA-binding protein involved in multiple steps of RNA metabolism and its expression is aberrant in breast cancer. Herein, we highlight novel implications of Sam68 in the mammary tumor onset and progression.

Material and methods: Tissue microarrays (TMA) were composed of n=155 histologically confirmed invasive breast carcinoma (T1-T2, N0, M0) and Sam68 expression was quantified as percent of immunoreactive tumor cells, a cut-off of 90% positive cells was chosen. Breast CICs were obtained from mechanically and enzimatically digestion of human breast tissues. Stable Sam68 knockdown was produced by lentiviral transduction and puromicin selection. Invasion assay was performed in 8µm pore size transwell coated with GF-depleted matrigel 1:3 in serum free medium. 3x104 cells were plated and 10% FBS medium was used as chemoattractant. Breast CICs (3 x 105) were suspended in 1:6 matrigel and orthotopically injected in NOD/SCID mice.

Results: Univariate analysis of the TMA data showed that invasive breast carcinoma over-expressing Sam68 (≥ 90% positive cells) were associated with a significantly higher incidence of distant relapse (P = 0.011). Notably, in a cohort of screened breast cancer patients, breast CICs expressed higher Sam68 protein levels than tumor bulk, highlighting that Sam68 expression was likely restricted to cells with self-renewal activity. Stable knockdown of Sam68 in breast CICs significantly curtailed proliferation rate and was coupled with an increase in p27kip1 and reduction of Bcl-xL. Compared to control cells, downregulation of Sam68 was correlated with an attenuation of cell motility capability and Twist, Snail, CD44v6 and Met levels. Moreover, Sam68 modulated the expression of an oncogenic alternative splice variant of Met (Met-SM), probably through the ASF/SF2 splicing factor. In addition, depletion of Sam68 sensitized breast CICs to standard chemotherapy. Finally, Sam68 silencing ablated breast cancer xenograft formation in immunocompromised mice.

Conclusions: Based on these results, we deem that Sam68 may promote pro-invasive signals by inducing and modulating the alternative splicing of oncogenic variants of several genes, such as CD44 and Met. Thus, further investigations on signaling pathways affecting CICs self-renewal and invasiveness could represent a crucial key to improve selective cancer treatment.

Citation Format: Alice Turdo, Miriam Gaggianesi, Antonina Benfante, Mauro Piantelli, Matilde Todaro, Giorgio Stassi. Sam68 sustains self-renewal and invasiveness of breast cancer initiating cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3897. doi:10.1158/1538-7445.AM2014-3897