Abstract 218: MEOX-1 as a novel cancer stem cell target for treatment of trastuzumab-resistant Her2+ breast cancers
Authors: Sun Lichao , Burnett Joseph P. , Gasparyan Mari , Korkaya Hasan , Jiang Hui , Liu Yajing , Connarn Jamie , Wicha Max , Sun Duxin 
Published: May 2015

Introduction: Previous studies showed that 60-80% of Her2+ breast cancers develop Trastuzumab-resistance after one year treatment and more than 50% of Her2+ Trastuzumab-resistant breast cancers have PTEN deletion and increased populations of cancer stem cells (CSCs). The purpose of this study is to explore novel targets to regulate cancer stem cells and to test treatment option for Her2+ Trastuzumab-resistant breast cancer.

Method: Trastuzumab resistance of Her2+ breast cancer cells (BT474) was induced by shRNA knockdown of PTEN and long term treatment of Trastuzumab (LTT) in comparison with Trastuzumab-sensitive BT474. The whole transcriptome RNA sequencing was performed and the differentially expressed genes were evaluated using dChip analyzer and the bioinformatics tool DAVID. Colony formation in soft agar and mammosphere formation were used to evaluate the effect of candidate genes on breast CSCs. Immunohistochemistry (IHC) was used to assess the protein expression levels in xenograft animal tissues and human breast cancer tissues. MTS assay was used to evaluate the effect of drug treatment to inhibit proliferation of BT474 and LTT. A xenograft model was used to determine whether drug efficacy to inhibit tumor formation. The secondary implantation was used to monitor cancer stem cells growth in vivo after treatment.

Result:

In comparison of BT474 and LTT under treatment of Sulforaphane (2-10 uM, 8-24 hr), RNA-Seq analysis revealed that 51 genes, including 23 up-regulated and 28 down-regulated by Sulforaphane treatment. DAVID functional clustering analysis revealed that 6 significantly altered genes were associated with 9 distinct functional categories. Among them, MEOX-1 was identified as top candidate to regulate Trastuzumab-resistance phenotype. siRNA knockdown of MEOX-1 in LTT cells inhibited mammosphere formation by 60.4% and colony formation by 86.7%. Further, in the IHC of 68 primary breast cancer samples, expression of MEOX-1 was positive in 34 primary breast cancer tissues (34/68, 50%), especially in triple negative breast cancer tissues (P&lt;0.05).

Sulforaphane inhibited the proliferation of LTT cells by 20% to 80%, while it showed no effect on BT474 cells in vitro. Also Sulforaphane decreased MEOX-1 mRNA level by 6-fold LTT cells. Daily injection with 50 mg/kg Sulforaphane reduced the growth of mouse xenografts by &gt;50% in SCID xenograft tumors (P&lt;0.05) and abrogated tumor growth after the reimplantation of primary tumor cells into the secondary mice (P&lt;0.05). Immunostainng showed that MEOX1 protein level in nucleus, and Sulforaphane significantly reduced MEOX-1 expression in primary xenograft tumors.

Conclusion:

MEOX1 is a novel target to regulate cancer stem cells in Her2+ Trastuzumab-resistant breast cancer cells. Drug treatment to down-regulate Meox1 provides treatment option for Trastuzumab-resistant breast cancer by eliminating breast cancer stem cells.

Citation Format: Lichao Sun, Joseph P. Burnett, Mari Gasparyan, Hasan Korkaya, Hui Jiang, Yajing Liu, Jamie Connarn, Max Wicha, Duxin Sun. MEOX-1 as a novel cancer stem cell target for treatment of trastuzumab-resistant Her2+ breast cancers. [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 218. doi:10.1158/1538-7445.AM2014-218