Metformin represses stem cell properties and induces apoptosis in human breast cancer cell and mouse mammary epithelial cell

강은주 고려대학교 대학원 2013 국내박사

Cancer stem cells (CSCs) are small portions of cancer cells that have important self-renewal and proliferation characteristics, and therefore, they play a significant role in cancer development and metastasis. Recently, several studies have revealed CSCs of several solid tumors. The identification and targeting of CSCs are expected to advance treatment outcomes, but until now, only a few drugs that specifically target CSCs have been developed. Metformin, an antidiabetic drug used for the treatment of type 2 diabetes mellitus, suppresses gluconeogenesis in the liver and increases insulin sensitivity and glucose uptake in peripheral tissues such as muscles. Moreover, the drug has shown an anti-cancer effect by inhibiting cellular proliferation and protein synthesis in several cancer cell lines. Additionally, many clinical and epidemiological studies have suggested that metformin might be associated with a decreased risk of cancer development and an increased response to chemotherapy. In addition to its anticarcinogenic effect, studies about the anti-cancer stem cell effect of metformin have been recently reported. Several reports revealed that metformin could preferentially kill cancer stem cells, including breast cancer stem cells. However, the mechanism of anti-cancer stem cell activity is not well known. Therefore, this study was planned to confirm the cytotoxicity of metformin, especially for breast cancer stem cells as well as breast cancer cells. To this end, several human breast cancer cell lines and normal mouse mammary epithelial cell lines were used. In this study, metformin showed cell growth inhibition in human breast cancer cells and mouse mammary epithelial cells by inducing cell cycle arrest and apoptosis. Moreover, metformin specifically killed breast cancer stem cells. Metformin showed reduced mammosphere formation in all cell lines. In addition, decreasing breast cancer stem cell population by metformin treatment was identified through FACS analysis. For the mechanism of anti-cancer stem cell activity, western blot assay was performed. We identified decreased levels of cyclin D and cyclin E and increased levels of Bax in secondary mammaospheres according to metformin treatment. In conclusion, this present study show that metformin kills breast cancer stem cells as well as breast cancer cells, and these phenomena occur by the induction of cell cycle arrest and apoptosis.

PC-3 유래 암줄기세포 유사세포의 분화에 영향을 미치는 약리활성 물질의 작용기전 규명

박주연 고려대학교 대학원 2020 국내석사

The incidence of male prostate cancer is not only the highest in the West except for skin cancer, but also a concern in Asia as the incidence is rapidly increasing due to changes in food and lifestyle. As with many other types of cancer, recurrent or metastatic prostate cancer also has a strong resistance to anticancer drugs, and in recent decades significant progress has been made in understanding the role of the Cancer Stem cell (CSC) in developing cancer resistance to chemotherapy or radiation therapy. By using a human-derived cancer cell PC-3 (prostate cancer cell)-derived cancer stem cell-like cells (CSC-like cells) established through preliminary research, It was intended to discover substances that can control the properties of cancer stem cells and to identify the mechanism of cancer stem cell control of the discovered materials. With this purpose, two main aspects were taken. First, to determine what roles dopamine receptor subtypes (DRD1, DRD2, DRD3, DRD4, DRD5) play in cancer stem cell-like cells, knockdown of receptors using siRNA for each receptor subtype and each dopamine receptor Inhibition of receptor activity through subtype specific antagonist treatment was performed. As a result, it was confirmed that inhibition of dopamine receptors D2 and D4 induces differentiation of cancer stem cells-like cells and reduces proliferation, migration and invasion. Second, the effect of each stereoisomer of crizotinib, known as a non-small cell lung cancer target treatment anticancer agent, on cancer stem cells-like cells was compared and analyzed. Unlike (S)-crizotinib, (R)- crizotinib not only inhibits the cell proliferation capacity of cancer stem cells-like cells, but also significantly changes its morphology and increases the cell size by about 2 times than the control group. Given that the phosphorylation of AMPK is selectively inhibited along with changes in the properties of cancer stem cell-like cells by (R)-crizotinib, it is believed that the combined suppression of these signaling materials is probably associated with the differentiation of cancer stem cell-like cells. While the survival rate of large numbers of cancer patients is increasing as early diagnosis and treatment of cancer are improved, many patients are still unable to be free from the fear of recurrence and metastasis that occurs after chemotherapy. Therefore, there is a need to develop a therapy capable of losing the characteristics of cancer stem cells. In this study, we proposed that the inhibition of dopamine receptor D2 and the inhibition of phosphorylation of AMPK may be related to the loss of characteristics of cancer stem cell-like cells. Therefore, further studies suggest that new regulatory substances and methods can be derived to regulate the characteristics of cancer stem cells if the mechanism of action directly related to the loss of characteristics of cancer stem cell-like cells is more clearly identified. 남성 전립선암의 발병률은 피부암을 제외하고 서양에서 가장 높을 뿐만 아니라, 식품과 생활양식의 변화로 인해 급격히 증가하고 있기 때문에 아시아에서도 우려의 대상이 되고 있다. 많은 다른 종류의 암의 경우처럼 재발되거나 전이된 전립선암은 항암제에도 강한 내성이 있는데, 최근 몇 십 년 동안 항암제나 방사선치료에 대한 암 저항성 발현에 있어 암줄기세포의 역할을 이해하는 데 상당한 진전이 있었다. 사전 연구를 통해 확립된 인체유래 전립선암세포인, PC-3유래 암줄기세포 유사세포(PC-3-derived cancer stem cell-like cells)를 이용하여 암줄기세포의 특성을 조절할 수 있는 물질을 발굴하고 발굴된 물질의 암줄기세포 조절 작용기전을 규명하 고자 하였다. 이러한 목적을 가지고 크게 두 가지 측면으로 접근을 하였다. 첫 번째로, 도파민 수용체 아형들(DRD1, DRD2, DRD3, DRD4, DRD5)이 암줄기세포 유사세포에서 어떤 역할을 하는지 규명하기 위해 각 수용체 아형들에 대한 siRNA를 활용한 수용체의 발현감소 및 각 도파민 수용체 아형에 특이적인 길항제 처리를 통한 수용체 활성의 억제 실험을 통해, 도파민 수용체 D2및 D4의 억제가 암줄기세포 유사세포의 분화를 유도하고, 증식능, 이동능, 침윤능을 감소시킴을 확인하였다. 두 번째로, 비소세포폐암 항암제로 잘 알려진 crizotinib의 각각의 입체이성질체가 암줄기세포-유사세포에 미치는 영향을 비교 분석해 보았다. (S)-crizotinib과 달리 (R)-crizotinib은 암줄기세포-유사세포의 세포증식능을 억제할 뿐 아니라 형태를 크게 변화시키며 세포 크기도 대조군보다 약 2배 정도로 증가시켰다. (R)-crizotinib에 의한 암줄기세포 유사세포의 특성 변화와 동반하여, AMPK인산화가 선택적으로 억제되는 것으로 볼 때, 이들 신호전달물질의 복합적 억제가 아마도 암줄기세포 유사세포의 분화와 연관될 것으로 판단된다. 암의 조기진단과 치료법이 향상되면서 암환자들의 생존율이 높아지고 있지만, 아직도 많은 환자들은 항암치료 후에 나타나는 재발, 전이의 공포로부터 자유로울 수가 없다. 따라서 암줄기세포의 특성을 상실시킬 수 있는 치료법의 개발이 더욱 필요한 실정이다. 본 연구에서 도파민 수용체 D2의 억제와 AMPK의 인산화 억제가 암줄기세포 유사세포의 특성 상실과 관련될 수 있다는 결과들을 제시하였기 때문에, 추가적인 연구를 통해서 암줄기세포-유사세포의 특성 상실과 직접적으로 연관되는 작용 기전이 보다 명확히 규명하게 된다면, 암줄기세포의 특성을 조절할 수 있는 새로운 조절물질 및 방법이 도출될 수 있을 것으로 사료된다.

Development of cancer stem cell-targeted therapy against breast cancer and its underlying mechanisms

김란주 강원대학교 대학원 2013 국내박사

1. Ras activation contributes to the maintenance and expansion of Sca-1pos cells in mouse model of breast cancer The cancer stem cell (CSC) hypothesis proposes that CSCs are the root of cancer and cause cancer metastasis and recurrence. The present study investigated whether Ras signaling is associated with stemness of the CSCs population characterized by the stem cell antigen (Sca-1) phenotype in a 4T1 syngeneic mouse model of breast cancer. The Sca-1pos putative CSCs had high levels of activated Ras and phosphorylated MEK (p-MEK), compared with counterparts. The Ras farnesylation inhibitor (FTI-277) suppressed the maintenance and expansion of CSCs. Therefore, selective inhibition of Ras activation may be useful for stem-specific cancer therapy. 2. High aldehyde dehydrogenase activity enhances stem cell features in breast cancer cells by activating hypoxia-inducible factor-2α High aldehyde dehydrogenase (ALDH) activity has been recognized as a marker of cancer stem cells (CSCs) in breast cancer. The present study investigated whether inhibition of ALDH activity suppresses stem-like cell properties in a 4T1 syngeneic mouse model of breast cancer. It was found that ALDH-positive 4T1 cells showed stem cell-like properties in vitro and in vivo. Blockade of ALDH activity reduced the growth of CSCs in breast cancer cell lines. Treatment of mice with the ALDH inhibitor diethylaminobenzaldehyde (DEAB) significantly suppressed 4T1 cell metastasis to the lung. Recent evidence suggests that ALDH affects the response of stem cells to hypoxia; therefore, the present study aimed to elucidate a possible link between ALDH and hypoxia signaling in breast cancer. Hypoxia-inducible factor-2α(HIF-2α) was highly dysregulated in ALDH-positive 4T1 cells. It was found that ALDH was highly correlated with the HIF-2α expression in breast cancer cell lines and tissues. DEAB treatment of breast cancer cells reduced the expression of HIF-2α in vitro. In addition, reduction of HIF-2α expression suppressed in vitro self-renewal ability and in vivo tumor initiation in ALDH-positive 4T1 cells. Therefore, these findings may provide the evidence necessary for exploring a new strategy in the treatment of breast cancer.

Regulation of Stemness Genes Involved in Functions of Embryonic Stem Cells and Cancer Stem Cells

Lee, Jina The Graduate School, Yonsei University 2019 국내박사

Embryonic stem cells (ESCs) derived from the inner cell mass of blastocysts are pluripotent and capable of self-renewal. Core transcription factors including Oct4, Sox2, and Nanog are prerequisites for maintaining stemness in ESCs. These factors should be repressed during stem cell differentiation. ESCs share the stemness properties of cancer stem cells (CSCs). CSCs are important in various key oncogenic properties, such as metastasis, chemoresistance, and tumor initiation. Although patients with liver cancer are generally treated with therapies that include chemotherapy and surgical resection, their prognosis is often poor because of cancer recurrence. In liver cancer, accumulating evidence suggests that the presence of CSCs may contribute to recurrence. Thus, targeting CSCs may be a therapeutic opportunity to manage liver cancer. This work aimed to study the regulation of stemness genes involved in functions of ESCs and CSCs. Tousled-like kinase 1 (Tlk1) is a serine/threonine kinase that is important for chromatin assembly, DNA repair, and cell cycle progression. In Caenorhabditis elegans and Drosophila, TLK depletion causes a developmental defect, but the role of Tlk1 in mouse ESCs (mESCs) is unclear. In this work, I generated Tlk1-knockdown and inducible Tlk1-overexpressing mESC lines and investigated the role of Tlk1 in the control of pluripotency and differentiation. Tlk1 depletion disrupted differentiation with delayed repression of core pluripotency factors. Conversely, Tlk1 overexpression caused downregulation of key pluripotency factors and induced differentiation with low alkaline phosphatase (AP) activity and a flattened morphology, even in the presence of leukemia inhibitory factor. Moreover, the forced expression of Tlk1 causes growth defect with an increase in the population of cells in G2/M phase. The data suggest that Tlk1 is essential for differentiation in mESCs. Embryonal carcinoma (EC) cells are considered the malignant counterparts of ESCs. EC cells have unique properties similar to those of CSCs. Thus, they can be used as a surrogate model for the study of CSCs. Calmidazolium chloride (CMZ) is a calmodulin antagonist and calcium channel protein inhibitor. CMZ can increase intracellular calcium concentration, which influences cellular processes that include apoptosis in various cell lines. The effects of CMZ on CSCs are unknown. I investigated the effects of CMZ on the EC cell line F9 as a surrogate model of CSCs. The CCK viability assay showed that CMZ inhibited the growth of F9 cells. CMZ induced apoptosis of F9 cells in association with caspase-3 activation and an increase in the sub-G1 cell population. RT-qPCR revealed that expression of pluripotency genes was down-regulated in CMZ-treated F9 cells. The collective results show that CMZ can inhibit the growth of EC cells by inducing apoptosis and down-regulating stemness-related genes. The third aim of the study was to examine the effects of JIB-04, a pan-inhibitor of Jumonji demethylases, on liver CSCs. Previous studies demonstrated anticancer activity of JIB-04 in lung cancer cells and prostate cancer cells. Also, my lab has reported that JIB-04 has selective anti-CSCs activity in colorectal cancer cells. Presently, downregulation of CSC-related genes and reduced tumorsphere formation ability were observed in liver cancer cells treated with JIB-04 compared to mock-treated cancer cells. The results suggest that JIB-04 may control liver cancer stemness. Collectively, this thesis research aimed to understand the control mechanisms of stemness, a critical and common feature in both ESCs and CSCs. The role of Tlk1 in the control of pluripotency and differentiation in ESCs has been demonstrated. The data indicate the potential activity of CMZ to selectively target EC, a surrogate model of CSCs. Finally, JIB-04 may be a potent agent to target CSC-like cells of liver cancer.

Role of p53 in transcriptional regulation of cancer stem cell marker CD133

박선영 성균관대학교 일반대학원 2011 국내석사

Cancer stem cell that was discovered in mouse acute leukemia model in 1937 has characteristics of cancer cell regulated poorly and normal stem cell capable of self-renewal and differentiation at the same time. Cancer stem cell theory suggested that cancer stem cell with tumor initiating and relapsing ability exists in cancer. It also insisted that we need to target cancer stem cell for complete anticancer therapy. Mechanism regulating these cancer stem cells contains to remove either Stemness gene or cancer stem cell marker uniquely expressed in cancer stem cell. p53 is a well-known tumor suppressor and has been reported to regulate self-renewal, differentiation, apoptosis of cancer stem cell as well as normal stem cell and cancer cell. Here, we studied the relations between p53 and cancer stem cell markers. The result shows that expression pattern of CD133, one of the cancer stem cell markers, is related with p53. CD133 is cell surface protein expressed in stem cell and cancer cell, which was usually used to sort cancer stem cell population into tumor cells as important cancer stem cell marker. Recently many researchers have presented that CD133 involved in tumor formation and metastasis and differentiation repression. When we compared with endogeneous p53 and CD133 expression level in each of cells that have wild type p53, mutated p53 and None, those levels were anti-parallel to each other. Furthermore, when we treated Doxorubicin(p53 inducing small molecule) into U2OS(wild type p53), NCCIT(mutated p53), H1299(none) and transfected pCDNA-HA tagging wild type p53 with H1299 in vitro, both protein and RNA level of CD133 certainly decreased. Consequently we carried out CD133 promoter analysis (Luciferase assay, ChIP) to indentify whether p53 suppresses CD133 expression level directly or not. As seeing results, Binding of p53 and H3K27 trimethylation (epigenetic repression marker) to CD133 promoters(P1, P2, P3, P4, P5) increased by overexpression of p53 in H1299 cell. It was equal when we treated doxorubicin in U2OS and MCF7 cells. Altogether these data suggest that p53 may regulate CD133 transcription by directly binding to the CD133 promoter. 암 줄기세포는 조절기전이 파괴되어 무한 증식하는 암세포와 자가생성능과 분화능을 가진 줄기세포의 특성을 둘 다 가지는 세포로, 1937년에 급성백혈병에서 종양형성가능세포로서 처음 발견되었다. 암 줄기세포 이론에 따르면 암 줄기세포만이 암 형성과 재발을 초래할 수 있기 때문에 근본적인 항암치료를 위해서는 암 줄기세포를 타겟으로 한 치료가 필요하다고 말한다. 이러한 암 줄기세포의 종양형성성을 억제하기 위해서 줄기세포유전자(Stemness gene)를 제거하는 방법과 암 줄기세포에 특이적으로 발현하고 있는 마커 단백질을 제거하는 방법이 연구되고 있다. p53은 잘 알려진 tumor suppressor로, 정상적인 줄기세포뿐만 아니라 암 줄기세포에서도 세포의 자가생성능 억제, 세포분화, 그리고 자가소멸을 조절하는 역할을 한다고 알려져 있다. 본 연구에서는 다양한 암 줄기세포 마커들을 대상으로 p53과의 관련성을 살펴보았고 그 결과, CD133의 발현양상이 p53에 의해 영향을 받고 있음을 관찰할 수 있었다. CD133은 세포표면에 존재하고 있는 세포표면단백질로 암세포나 줄기세포에서 발현하여 암 조직의 형성이나 전이, 그리고 줄기세포의 분화억제 등에 관여하는 대표적인 암 줄기세포 마커로서 연구되고 있다. 각각 Wild type p53, Mutated p53, None p53을 가진 여러 종류의 암세포와 암 줄기세포에서 endogeneous p53과 CD133의 발현 양을 비교하였을 때, p53이 많이 발현하고 있을수록 CD133의 양은 감소되어 있다는 것을 확인하였다. p53 발현을 유도한다고 알려진 small molecule인 Doxorubicin을 U2OS(Wild type p53), NCCIT(Mutated p53), H1299(None p53) cell에 처리하거나, HA tagging wild p53을 H1299 cell에 과 발현시켰을 때 모두 p53이 증가됨에 따라 CD133의 단백질과 RNA양이 감소되었다. p53이 CD133의 발현을 억제하고 있는 것이 직접적인 p53의 전사조절 때문인지 알아보기 위해서 p53을 대상으로 CD133 promoter분석을 시행하였다. p53의 양이 증가함에 따라 CD133 Promoter에 p53의 결합 정도가 확연히 증가되었다. 이때, 후성유전체학적 억제마커인 3번 히스톤 27번째 라이신(H3K27)의 Trimethylation도 함께 증가된 것으로 보아 p53에 의해 CD133의 전사가 억제되었음을 알 수 있었다. 이 모든 것을 종합하였을 때, p53은 CD133의 promoter에 직접적으로 결합하고 있고 이를 통해 CD133의 전사활성화를 억제하고 있다는 것을 알 수 있었다.

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

윤효진 서울대학교 대학원 2019 국내박사

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.

Inhibition of PI3K and mTOR increases efficacy of paclitaxel in in vitro and in vivo colorectal cancer stem model

구정은 College of Life Sciences and Biotechnology, Korea 2014 국내석사

This study focused on the signal pathways of Phosphatidylinositol 3 kinase (PI3K) and mammalian target of Rapamycin (mTOR) which are regulator of proliferation, differentiation, cell growth and death in cancer cells. CD133 marker was used to isolate cancer stem cells and non-cancer stem cell which is a glycoprotein also known in humans and rodents as Prominin 1 (PROM1). It shows that inhibition of dual PI3K and mTOR signal pathways is more effective than single blockage of each pathway in cancer stem cells. And it reduced their stemness properties and increased differentiation in dual blocked CSCs. On the basis of these, it was experimented with balb/c nude mice to confirm the effect of drug sensitivity against paclitaxel (taxol) in vivo. And it shows the result of effect on dual blocking PI3K and mTOR signal pathway. Both blocking of mTOR and PI3K has lower cell viability than single blocking as well as decrease of tumor formation. And paclitaxel treatment also has more effective in dual-blocking, which was treated with NVP-BEZ235. Taken together, these results indicate that dual blocking PI3K and mTOR induced differentiation of cancer stem cells and it can be more effective on chemotherapy of cancer.

S100A14 regulates stemness in chemoresistant colon cancer cells by regulation of STAT3 stability

심정연 서울대학교 대학원 2021 국내석사

Colon cancer is the third most common cancer. Of the several therapies for colon cancer, chemotherapy has been used most frequently. 5-Flourouracil (5-Fu) and oxaliplatin are first-line chemotherapeutic agents used for treatment of colon cancer. Although these drugs are commonly used for therapy, several evidences have indicated that anti-cancer drugs induce chemoresistance, therefore reducing the effectiveness of treatment. Because of heterogeneity, cancer cells respond to treatments differently. Remarkably, the stem cell property of cancer cells has been reported to be highly correlated with chemoresistance. In this paper, when cancer cells become resistant to anticancer drugs, we investigate the changes in cell properties and internal cell signaling pathways Here, we generated drug resistant colon cancer cells after chronic exposure to 5-Fu and oxaliplatin. We found that resistant cells have highly upregulated stemness property. Through microarray and big data analysis, we found that S100A14 level was most decreased in drug resistant cells. Increased stemness property in resistant cells was reduced after S100A14 transfection. We also found that S100A14 level was decreased in colon cancer with high stem cell population. To investigate how S100A14 regulates stemness, we analyzed resistant cells using luciferase assay to find increased signaling pathway. STAT3 pathway was most upregulated in both cells. HCT116 resistant cells have reduced S100A14 expression and increased STAT3 expression. To determine the relation between S100A14 and STAT3, we analyzed STAT3 level in S100A14 modulated cells. STAT3 protein level was downregulated in S100A14 overexpression cells, while it was increased in S100A14 knockdown cells. Next, we investigated that S100A14 regulated STAT3 at a mRNA level or protein level. There are no significant changes in mRNA levels between parental cell and resistant cells. On the other hand, STAT3 proteins were rapidly degraded when S100A14 level was high; therefore, we have demonstrated S100A14 regulates STAT3 stability. Finally, we found that STAT3 stability was regulated by interaction with S100A14. Through immunoprecipitation, we confirmed that STAT3 degradation was increased because S100A14 facilitated binding of more ubiquitin to STAT3. These findings suggest novel function of S100A14 in regulation of stemness property in chemoresistant colon cancer. Moreover, S100A14 level can be used to estimate stem cell population in colon cancer. Thus, S100A14 can be a potential biomarker for chemotherapy of colon cancer 대장암은 전 세계적으로 세 번째로 흔한 암종으로 주요 사망 원인 중 하나이다. 대장암 치료에 사용되는 다양한 방법 중에서, 항암제를 이용하는 화학요법은 가장 많이 사용되어 진다. 5-플로오로 우라실 (5-Fluorouracil)과 옥살리플라틴 (oxaliplatin)은 대장암 치료에 있어 주로 사용되는 화학요법제이다. 이와 같은 약물은 치료에 있어 흔히 사용되지만, 다양한 연구들은 항암제가 오히려 약에 대한 저항성을 유도해 치료 효율을 낮춘다는 것을 보여주고 있다. 암세포 내부의 이질성 (heterogeneity)으로 인해, 암세포는 치료에 있어 다양한 반응성을 가지게 된다. 특히 암 줄기세포는 항암제 저항성과 높은 상관관계를 가진다는 것으로 보고되고 있다. 본 논문에서는 암세포가 항암제 저항성을 가지게 되었을 때, 세포의 성질 변화와 그리고 이를 유도하는 내부 기작을 밝히고자 하였다. 우선 대장암 치료에 많이 쓰이는 항암제인 5-플로오로우라실 (5-Fluorouracil)과 옥살리플라틴 (oxaliplatin)을 세포에 장기간 노출시켜 약물에 저항성을 가지는 세포를 구축하였다. 이렇게 만들어진 항암제 내성 세포는 증가된 줄기세포능 (Stemness)을 가진다는 것을 발견하였다. 그리고 구축된 세포 간의 마이크로어레이 실험을 통해 저항성을 가진 세포에서 변화하는 유전자를 분석하였고, 이를 통해 S100A14가 저항성 세포에서 가장 낮아져 있다는 것을 발견하였다. 저항성 세포에서 증가되어진 줄기세포능은 S100A14가 transfection될 경우 낮아졌으며, 대장암에서 줄기세포능을 많이 가지는 부분일수록 낮은 S100A14 발현을 보인다는 것을 확인하였다. S100A14가 어떻게 줄기세포능을 조절하는지 알아보기 위해서, 본 실험에서는 luciferase assay를 실행하여 항암제 저항성 세포에서 증가되어진 기작을 찾고자 하였다. 다양한 신호전달 경로 중에서 STAT3와 관련된 신호전달 기작이 대장암 항암제 저항성 세포에서 큰 폭으로 증가 되어있는 것을 확인하였다. 화학 항암제에 저항성을 가진 HCT116 세포는 낮은 S100A14 발현 보이며, 저항성이 없는 기존의 HCT116 세포에 비해 높은 수준의 STAT3를 가진다. S100A14와 STAT3 사이의 관계를 알아보기 위해, S100A14를 없애거나, 과발현시켜 양을 조절하였을 경우 STAT3가 달라지는지를 확인해 보았다. 저항성 세포에서 STAT3 단백질은 S100A14를 transfection할 경우 줄어들었으며, 반대로 S100A14를 없앤 세포에서는 STAT3 단백질이 증가하였다. 우리는 이러한 과정에서 S100A14가 STAT3를 mRNA 발현이 아닌 단백질 수준에서 조절한다는 것을 찾아냈고, S100A14가 STAT3 분해를 조절한다는 것을 알아냈다. S100A14가 어떻게 STAT3 분해를 조절하는지 알아보기 위해 면역침강실험을 진행하였고, 이를 통해 S100A14가 STAT3에 결합하여 STAT3의 유비퀴틴화 (ubiquitination)를 조절하여 STAT3 분해를 유도한다는 것을 밝혔다. 이 연구를 통해, 화학 항암제에 저항성을 보이는 대장암 세포에서 줄기세포능 조절에 있어서 S100A14의 새로운 기능을 규명하였다. S100A14은 STAT3의 분해를 조절하는데, 저항성 세포에서는 S100A14의 발현이 낮아져 STAT3가 증가하게 되고, 이로 인해 줄기세포능을 유도하는 여러 요소들이 증가하게 된다는 것을 본 논문을 통해 밝힌 것이다. 더 나아가, S100A14가 항암제 치료의 효과를 예측할 수 있는 바이오 마커로 사용될 수 있다는 점을 본 논문을 통해 제시하였다.

Analysis of ABC transporters and multidrug resistance of CD133+ cancer stem cells in colorectal cancer

이상한 Graduate School, Korea University 2015 국내석사

Recent researches reveal the existence of distinct minor group of cells in cancer. This group of cells was termed as cancer stem cells, where the term suggests, the sharing of similar characteristics with normal stem cells. The idea of cancer stem cell hypothesis is that cancer stem cells possess the properties of self-renewal, multilineage differentiation capacity and the essential property of self-protection through activity of ATP binding cassette transporter. The purpose of the study is to identify the relation between ATP transporter expression levels and multidrug resistance in both cancer stem cells and non-cancer stem cells. CD133 marker was used to isolate cancer stem cells and non-cancer stem cell. Multidrug resistance is enriched in CD133+ cancer stem cells compared to their CD133- counterpart. On the basis of these, this study was experimented with SW620 cells and HT29 cells to confirm the effect of drug resistance and cell viability. The results show that cell viability and drug resistance were enriched in CD133+ SW620 cancer stem cells compared to CD133- SW620. Moreover, ABC transporter expression levels increased in CD133+ cancer stem cells. On the other hands, cell viability and resistance to drugs were enriched in CD133+ HT29 cancer stem cells compared to their CD133- HT29 counterpart. However, ABC transporter expression levels almost never regulate in CD133+ cancer stem cells. And also, the CD133+ of all cell lines exhibited higher subtle multidrug resistance by ABC transporters than CD133-. These results indicate that not all cells possessing multidrug resistance have increased expression of ABC transporters. Multidrug resistance was associated not only ABC transporters but also the other.

Effect of thioridazine, an antipsychotic and dopamine receptor antagonist, on the differentiation of cancer stem cell-like cells derived from PC3 cells

Roney, Md Saiful Islam Graduate School, Korea University 2017 국내석사

ABSTRACT Over the last few decades, cancer stem cell (CSC) research has gained unprecedented attention and emerged as a promising tool in cancer treatment. Growing evidence suggests that the presence of CSCs in numerous cancer types is the root cause of drug resistance. Recent clues reveal that prostate cancer, the second most oftentimes diagnosed cancer around the world, contains CSCs and is not curative in case the disease outstretches to an advanced state. Failure in treating prostate cancer with current therapeutics has been linked to the presence of CSCs in its tumor mass. Cancer stem cell’s ability to withstand current chemotherapeutics and its contribution to tumor relapse has changed the paradigm of drug development, screening and cancer biology. Recently, dopamine receptor antagonists have been identified as potent anti-CSC agents in a number of cancers and inducers of CSC differentiation, a way of differentiating CSCs into different cell types which, by stymieing its stem cell properties, could sensitize them to existing chemotherapy. However, no study has demonstrated the effects of dopamine receptor antagonist against prostate cancer stem cells (PCSCs). This study focused on evaluating the effect of thioridazine, a potent dopamine receptor antagonist, on PCSCs, exploring the mechanism by which thioridazine induces differentiation of PCSCs. In our study, we observed that thioridazine-induced morphology changes of PC3 derived cancer stem cell-like cells. PCSCs expressed some well-known stem cell markers, such as Sox2, E-cadherin, Oct3/4, and Klf4 which were downregulated by thioridazine in a dose-dependent manner. Signal transduction molecules such as Akt and AMPK were also downregulated by thioridazine. Further study revealed that the inhibition of AMPK induced morphological changes, comparable to that of the changes in morphology induced by thioridazine, which could be reversed by AMPK activator dose-dependently. Based on our conclusive findings, we propose that thioridazine induces differentiation of prostate cancer stem cells (PCSCs) via AMPK downregulation, along with the attenuation of stemness and pluripotency attributes.