<Introduction>

Cancer stem cells (CSCs) have been defined by the potential to self-renew and to differentiate. CSCs pose a major hurdle in the treatment of cancer. However, the mechanisms by which cells acquire CSC properties such as drug resistance remain unclear.

Dual-specificity tyrosine-regulated kinase 2 (DYRK2) is a protein kinase that phosphorylates its substrates on serine/threonine. Initially, we found that DYRK2 phosphorylates p53 at Ser 46 to regulate apoptotic cell death in response to DNA damage. Recently, we have shown that DYRK2 controls Snail degradation in breast cancer and ovarian serous adenocarcinoma. We also found that knockdown of DYRK2 in luminal-type breast cancer MCF-7 cells increased the cancer stem cell population.

Kruppel-like factor 4 (KLF4) is one of the Yamanaka factors. It has been reported that pluripotent stem cells from mouse embryonic or adult fibroblasts are induced by introducing four factors, Oct3/4, Sox2, c-Myc, and Klf4. This finding led us to determine if KLF4 is indispensable for the maintenance of CSCs.

The aim of this study is to clarify whether DYRK2 regulates CSCs through KLF4 in breast cancer.

<Methods>

Cell lines: MCF-7 (human mammary carcinoma: ATCC) cells were grown according to standard protocols.

We established stable DYRK2-depleted cells. MCF-7 cells were transfected with pSuper-puro vector (pSuper control) or pSuper-puro DYRK2 shRNAs (shDYRK2) with puromycin to isolate stable cell lines.

In turn, we established both stable DYRK2- and KLF4-depleted cells. shDYRK2 cells were transfected with pSuper-neo vector (pSuper-neo control) or pSuper-neo KLF4 shRNAs (shKLF4) with puromycin and G418. Knockdown of DYRK2 or KLF4 was confirmed by real-time RT-PCR and immunoblotting.

The depleted cells were compared with the control cells using real-time RT-PCR, immunoblotting, flow cytometric analysis, mammosphere assay, xenograft models and immunohistological staining.

<Results>

We analyzed the population of breast cancer stem cells by flow cytometric analysis and in vitro mammosphere assay. The results showed that knockdown of DYRK2 was associated with the increase of CD44+/CD24- cells. While pSuper control cells formed mammospheres, they did in a lesser extent compared to shDYRK2 cells.

In real-time RT-PCR and immunoblotting analysis, stable DYRK2 depletion in MCF-7 cells induced KLF4 accumulation.

We then investigated the effect of KLF4 on stemness by flow cytometric analysis and in vitro mammosphere assay. The results showed that knockdown of KLF4 in shDYRK2 cells reduced the proportion of CD44+/CD24- cells. Whereas shDYRK2/shKLF4 cells formed mammospheres, they did in a lesser extent compared to shDYRK2/pSuper-neo control cells. Moreover, the scale of the mammospheres formed in shDYRK2/shKLF4 cells was significantly smaller, as compared with that in shDYRK2/pSuper-neo control cells.

In xenograft models, the loss of KLF4 protein expression significantly decreased tumor formation.

Immunohistological staining of fifty-nine samples from surgically treated breast cancer patients showed an inverse correlation between DYRK2 and KLF4 expression.

<Conclusion>

These findings revealed that DYRK2 contributes to the generation of breast cancer stem cells through KLF4.

Citation Format: Imawari Y, Mimoto RK, Yamaguchi N, Kamio M, Kato K, Nogi H, Toriumi Y, Uchida K, Takeyama H, Yoshida K. DYRK2 contributes to the generation of breast cancer stem cells through KLF4 [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-07-07.