Loss of Caveolin-1 promotes stem-like traits in breast cancer cells

Abstract

암줄기세포 (Cancer stem cell)는 종양의 개시 (initiation), 전이 (metastasis), 그리고 재발 (recurrence)에 있어 중요한 역할을 한다. 최근 연구 동향을 살펴보면, 암의 진행에 있어서 암줄기세포의 복잡한 생리학적 기능을 이해하고, 이들의 발생 기전을 조절하는 치료법 개발이 강조되고 있다. 본 연구에서는 그 발생 기전을 조절하는 조절인자 (modulator)로서 Caveolin-1 (Cav-1)을 선정하였다. Cav-1은 세포막 단백질인 Caveolae를 구성하는 주요 단백질로써, 세포내 콜레스테롤 항상성 유지 (cholesterol homeostasis), 분자 수송 (vesicle trafficking), 암의 진행 (tumor progression) 및 신호 전달 (signal transduction) 등 다양한 기능에 관여한다. 그러나 Cav-1의 암줄기세포에 대한 분자기전에 대한 연구는 미흡한 실정이다. 이에 본 연구에서는 암줄기세포의 발생기전에 있어서 Cav-1의 역할에 대해 알아 보고자 하였다. 먼저, 줄기세포 배양방법으로 잘 알려진 sphere-forming culture system을 통해 배양된 유방암 줄기세포 (tumorsphere MDA-MB-231)에서 Cav-1의 발현이 낮아지는 것을 확인하였다. 유방암세포 MDA-MB-231에 small interfering RNA (siRNA) 기법을 통해 Cav-1을 억제하였을 때, 줄기세포에서 많이 나타나는 특성인 자가재생 (self-renewal)과 epithelial-mesenchymal transition (EMT)의 표현형 (phenotype)이 증가하고, 더불어 구체형성능 (tumorsphere forming capacity)이 증가하는 것을 확인하였다. 구체형성 세포 (tumorsphere cell)에서 Cav-1의 발현이 낮아지는 것은 유비퀴틴화에 의한 단백질 분해 조절 기전임을 확인하였다. Cav-1의 활성에는 tyrosine 14번 잔기가 주로 역할을 하고 있다는 문헌 정보를 토대로, 특정 부위 돌연변이 (site-directed mutagenesis)를 통해 tyrosine 14번을 phenylalanine으로 치환하였을 경우, 자가재생능과 EMT 표현형이 감소되는 것을 통해, 줄기세포능을 유지하는데 있어서 Cav-1의 tyrosine 14 잔기가 매우 중요한 역할을 하는 것을 확인하였다. 또한 Cav-1이 다른 분자와 상호간의 직접적인 결합을 통해 신호 전달에 기여한다는 보고를 바탕으로 그 후보물질로서 Nrf2를 선정하여, 실제 이들이 유방암 세포에서 결합을 이루고 있음을 확인하였고, tumorsphere cell에서 Cav-1의 단백질 안정화가 감소되면서 Cav-1과 결합되어 있던 Nrf2가 핵 안으로 이동되어 줄기세포능을 증가시키는 것을 관찰하였다.

최근 암줄기세포를 타겟으로 하는 암의 치료 및 예방을 위해 비교적 독성이 약한 식물 유래 화합물 (phytochemicals)을 이용한 연구가 많이 이루어 지고 있다. Resveratrol은 그 가운데 대표적인 물질 중 하나로, 포도, 건과류 및 장과류 (berries) 등에 다량 함유되어 있는 폴리페놀 성분 물질로 항산화 효과, 심혈관계질환 예방효과, 항암효과, 염증관련 질환 예방 효과를 가진 것으로 보고되고 있다. Resveratrol을 처리하였을 때, 유방암세포의 구체형성능과 자가재생, EMT 표현형이 감소하고, Cav-1의 발현은 증가하는 것을 확인하였다. 이때 증가한 Cav-1을 siRNA를 이용하여 억제시키면, 감소하였던 줄기세포능이 다시 증가함을 확인하였다. 이와 같은 결과를 바탕으로 resveratrol이 Cav-1을 매개로 한 경로 조절을 통해 암줄기세포적 성격을 억제하여, 향후 암줄기세포를 타겟으로 하는 새로운 치료법의 접근이 가능할 것으로 사료된다.

Cancer stem cells (CSCs), a subset of cancer cells characterized by the property of self-renewal and differentiation, initiate tumor growth and promote chemo-/radiotherapy resistance, which are considered to be responsible for progression, recurrence and metastasis. Several intrinsic and extrinsic signaling pathways maintaining CSCs population have been explored. A prominent property of CSCs is the ability to undergo self-renewal division. The dysregulation of self-renewal signaling may account for the regeneration of the tumor. Hence, understanding the signaling pathways for maintaining features of CSCs is likely to be important for developing targeted anticancer therapies.

Caveolin-1 (Cav-1) is a major protein of caveolae, which is flask-shaped invagination at cell membranes. Caveolae participate in various cellular functions, such as vesicle trafficking, cholesterol homeostasis, and tumor progression. Cav-1 is thought to regulate the activity of proteins, such as Src family kinases, H-Ras, protein kinase C, epidermal growth factor tyrosine kinase, extracellular signal-regulated kinase, and endothelial nitric oxide synthase involved in oncogenic signaling pathways. In this context, Cav-1 has been proposed as a potential therapeutic target for disrupting tumor progression and metastasis.

In the present study, I investigated a role for Cav-1 in regulating the stemness of human breast cancer (MDA-MB-231) cells. To investigate whether Cav-1 could be involved in modulating the stemness of breast cancer cells, tumorspheres were generated from adherent cells. Cav-1 expression was significantly lower in tumorspheres than in adherent cells. Further, the proportion of breast stem-like CD44high and CD24low cells was increased in Cav-1 knocked down MDA-MB-231 cells. Mechanistically, the silencing of Cav-1 resulted in the elevated expression of the well-known stemness-related genes, Nanog, Oct 3/4, and Sox2 with concomitant upregulation of Bmi-1, a representative self-renewal regulator. In line with above findings, the Cav-1 knock down increased the size and the number of spheres derived from MDA-MB-231 cells. In a CSC-like state, the reduced Cav-1 levels were attributable to its destabilization through ubiquitin-proteasome degradation. Src-mediated phosphorylation of Cav-1 at the Tyr 14 residue is essential for its degradation. The expression of p-Cav-1 (Tyr14) and p-Src (Tyr416) was markedly elevated in tumorsphere cells, compared to adherent cells. Y14F Cav-1 mutation by replacing Tyr14 with phenylalanine was sufficient to abolish phosphorylation and ubiquitination of Cav-1 protein. To verify that Cav-1 suppresses the breast cancer cell stemness, we overexpressed Cav-1 in MDA-MB-231 tumorspheres. As a result, the MDA-MB-231 tumorspheres overexpressing WT-Cav-1 exhibited the reduced ability of spheroid formation compared with Mock treated control cells. Notably, the tumorsphere-forming ability of cells expressing Y14F mutant Cav-1 was weaker than that of cells expressing WT-Cav-1. Likewise, the Y14F Cav-1 mutation significantly mitigated the inhibitory effects of Cav-1 on the expression of Bmi-1 and EMT markers. Taken together, these findings suggest that Cav-1destabilization by Src-mediated phosphorylation may play a pivotal role in manifestation and maintenance of stemness in breast cancer cells.

However, a question that still needs to be addressed is how Cav-1 destabilization promotes stemness properties. The Nuclear erythroid 2 p45-related factor-2 (Nrf2) is a key transcription factor that regulates the antioxidant and detoxification enzymes. Recently, it has been reported that Nrf2 signaling is involved in CSC-like properties of several types of cancer cells. Notably, Nrf2 has been reported to directly interact with Cav-1. In breast cancer, Cav-1 binds to Nrf2 and enhances its degradation in cytosol through ubiquitination. The reduction of the intracellular accumulation of Nrf2 by Cav-1 led to a decrease in stemness-related gene expression. When degradation of Cav-1 occurs through ubiquitin-proteasomal degradation, Nrf2 is dissociated from the Cav-1 complex. The liberated Nrf2 accumulates in the nucleus and enhances the expression of stemness-related genes, which promote the manifestation of CSC-like phenotypes. Thus, Cav-1 interact with Nrf2 in breast cancer and inhibits the Nrf2 signaling, thereby suppressing the manifestation of CSC-like properties.

Recently, natural products have attracted much attention for prevention and treatment of cancer. Resveratrol (trans-3,4,5-triydroxystilbene), a phytoalexin found in grapes and other food products, has been investigated with regard to its chemopreventive and chemotherapeutic activities in various cancer cells as well as in animal models. Nonetheless, the underlying mechanism by which resveratrol regulates the signal transduction pathway involved in maintenance of CSCs and manifestation of their characteristics still remains to be largely unclear. Therefore, I investigated the effects of the resveratrol on breast cancer stem-like properties in the context of its modulation of Cav-1-mediated signaling. In this study, I found the inhibitory effect of resveratrol on the viability and migration capacity of tumorsphere MDA-MB-231 cells. Further, resveratrol significantly decreased the number and the size of MDA-MB-231 tumorspheres and also the proportion of CD44high/CD24low cell population Mechanically, the expression of self-renewal signaling molecules and EMT markers was reduced by treatment of resveratrol. Notably, resveratrol treatment decreased expression of self-renewal and EMT markers, which was elevated by knockdown of Cav-1.

In conclusion, reduced Cav-1 accumulation depends on its destabilization through ubiquitin-proteasome degradation. After degradation of Cav-1, Nrf2 is dissociated from the Cav-1 complex and enhances the expression of stemness-related genes.. Resveratrol has an inhibitory effects on manifestation of Cav-1-mediated CSC-like properties. This study herein indicates that Cav-1 acts as a pivotal regulator in manifestation and maintenance of stemness in breast cancer cells and can be an important therapeutic target for breast cancer therapy.