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Project Details
Funding Scheme : General Research Fund
Project Number : 782111
Project Title(English) : Unraveling the functional roles and underlying mechanisms of p70 S6 kinase in multicellular spheroid formation and as a regulator of N-cadherin 
Project Title(Chinese) : 解開p70S6K在多細胞聚合過程及調控N-cadherin中的角色及機制 
Principal Investigator(English) : Prof Wong, Alice Sze-Tsai 
Principal Investigator(Chinese) :  
Department : School of Biological Sciences
Institution : The University of Hong Kong
E-mail Address : awong1@hku.hk 
Tel : 22990865 
Co - Investigator(s) :
Prof YAM, Judy Wai Ping
Panel : Biology & Medicine
Subject Area : Biological Sciences
Exercise Year : 2011 / 12
Fund Approved : 980,000
Project Status : Completed
Completion Date : 30-11-2014
Project Objectives :
To study the cellular events modulated by N-cadherin with a view to elucidate its pro-metastatic role in p70S6K-expressing cells
To unravel the underlying mechanisms of action of N-cadherin with particular reference to its potential role(s) in the regulation of catenins, receptor tyrosine kinases, and non-canonical Wnt signaling
To decipher the mechanisms by which N-cadherin is regulated by p70S6K
Abstract as per original application
(English/Chinese):
大約90%的癌症死亡個案是由腫瘤轉移引致的，因此，明白腫瘤轉移的機制非常重要。在腫瘤轉移的過程中，癌細胞要在沒有附着的環境下存活的先決條件是能夠形成多細胞聚合(Multicellular aggregates/spheroids, MCS)。但到目前為止，對控制MCS形成過程的因素仍所知甚少，越來越多證據顯示微環境中的信號在癌症演進中非常重要。卵巢癌是一種極具轉移能力的癌症，透過腹膜散播和產生腹水而提供一個充滿各種可溶性生長因子的微環境予癌細胞，我們已於早前證明了這些生長因子對細胞的活動有重大的影響，而於最近，我們的實驗結果更顯示p70S6K，一個磷脂酰醇3-激酶/AKT的下游受應器, 在細胞內扮演着信號傳遞的角色，協調多個生長因子的作用。此外，我們亦首次證實p70S6K 除了控制細胞分裂和生長外，更可涉及於其他腫瘤演進的關鍵過程，包括遠端轉移。我們已經建立了一個三維的受體外模型以模擬癌細胞在體內沒有附着的生長環境，希望藉此發現及建構出腫瘤轉移的機制。我們的實驗顯示p70S6K在MCS的形成過程是關鍵的調節物。再者，N-cadherin的高表達在此過程中看來是必須的，而這細胞間的黏附分子通常在較後期的癌症及腹膜性轉移才出現，由此，我們猜想p70S6K可能透過增加MCS的形成和散播而在卵巢癌的早期演進有着關鍵性的作用，我們更猜想MCS的形成和散播可能由N-cadherin作傳遞或管制媒介， 在此研究中，我們會(i)研究N-cadherin在細胞內所介導的事項，以建立其於p70S6K表達細胞中誘導腫瘤轉移的角色; (ii)闡明其隱含機制，特別是N-cadherin在調節連結蛋白，受體酪氨酸蛋白激及非典Wnt信號; 及(iii)解釋p70S6K調控N-cadherin的機制。這個研究將令我們更清楚MCS在腫瘤轉移的角色及機制及為研制新的醫療方法提供方向。
Realisation of objectives: Objective 1: Multicellular spheroid (MCS) is a critical feature of ovarian cancer whose adhesive abilities have not been elucidated. During the past years, using a 3-dimensional culture, we have successfully shown for the first time that p70S6K plays a pivotal role in the formation of ovarian carcinoma MCS, which is mediated by N-cadherin, and suggest that the subsequent adhesion to the peritoneal mesothelium, which is the first rate-limiting step in ovarian cancer metastasis, is a P-cadherin-dependent event. We also present evidence that a novel interplay linking P-cadherin to regulation of b1 integrin activation via an induction of the Golgi glycosyltransferase ST6Gal-I, which mediates b1 integrin hypersialyation in this process. Percentage achieved: 100% Objective 2: Despite its significance, the key signals and molecular events regulating the formation and expansion of MCS remain elusive. Our results show that N-cadherin has a dominant effect over other cadherins on MCS formation of ovarian cancer cells. In addition to cell-cell adhesion, we show that N-cadherin can activate specific downstream signal transduction. We identify a novel role for N-cadherin in non-canonical Wnt/Ca2+ pathway. Furthermore, whereas b-catenin and Ca2+ can transduce the non-canonical Wnt signaling pathway independently, we show that N-cadherin acts via b-catenin and Ca2+ in a coordinated interdependent manner, suggesting that crosstalk in Wnt/b-catenin and Wnt/Ca2+ activation as a mechanism of N-cadherin and MCS formation. Percentage achieved: 100% Objective 3: In search of how p70S6K may regulate N-cadherin, we show that this is mediated through activation of the transcription factors Twist and Sox9. Interestingly, we found that this upregulation of Twist and Sox9 is not the result from changes in rate of transcription, but mRNA stabilities. Twist and Sox9 do not epistatically interact and more likely be each have an essential role in regulating N-cadherin expression. Using computational and experimental approaches, we thus identify a role of miR-145 in coregulating both Twist and Sox-9 to enhance N-cadherin expression due to p70S6K activation. We also provide mechanistic insights suggesting that this tumorigenic activity is associated with the ability of p70S6K to regulate miRNA biogenesis through a Dicer-tristetraprolin interaction. Percentage achieved: 100% Journal Publications: 1. Ip and Wong. Expert Opin. Ther. Targets 16: 619-630 (2012) 2. Ip et al., Oncotarget 5: 9133-9149 (2014) 3. Kala et al., J. Med. Chem. 57: 2634-2642 (2014) 4. Lam et al., Cancer Res. (submitted) (2015) 5. Lam et al., in preparation (2015) Book chapter: 6. Ip et al., In: Cancer Therapy: Recent Progress and Future Perspectives, Bentham Science Publishers [Invited]
Summary of objectives addressed:
Objectives Addressed Percentage achieved
1.To study the cellular events modulated by N-cadherin with a view to elucidate its pro-metastatic role in p70S6K-expressing cellsYes100%
2.To unravel the underlying mechanisms of action of N-cadherin with particular reference to its potential role(s) in the regulation of catenins, receptor tyrosine kinases, and non-canonical Wnt signalingYes100%
3.To decipher the mechanisms by which N-cadherin is regulated by p70S6KYes100%
Research Outcome
Major findings and research outcome: Objective 1: Using a 3-dimensional culture, we have identified for the first time that p70S6K is an important regulator for the seeding and metastatic spread of MCS on the peritoneum. We further demonstrated that two mechanisms are operated in sequence: the initial requirement of N-cadherin in the formation of MCS, and the later involvement of P-cadherin in the subsequent adhesion to the peritoneal mesothelium, which is in support of the sequential steps of the ovarian metastatic cascade. Moreover, we provide evidence of a novel interplay linking P-cadherin to the regulation of b1 integrin activation via the induction of the Golgi glycosyltransferase ST6Gal-I in this process. While these are seen as distinct systems, there is increasing evidence that a fine-tuned crosstalk between cadherins and integrins can generate a high degree of specificity in cell adhesion than the intrinsic specificity of individual receptors. Publications: Journal article: #2 Conference proceeding: #1 [awarded ECCO Fellowship Grant] Objective 2: We show for the first time that N-cadherin has a dominant effect over other cadherins on MCS formation of ovarian cancer cells. In addition to cell-cell adhesion, we show that N-cadherin can activate specific downstream signal transduction via Wnt/b-catenin and Wnt/Ca2+ crosstalk. This may indicate how an increase in N-cadherin expression in MCS is likely to promote the progression of ovarian cancer. Journal article: #2, #5 Objective 3: On exploring how p70S6K may regulate N-cadherin, we show that this is activated through two critical transcriptional regulators Twist and Sox9. Interestingly, we found that this activation of Twist and Sox9 is not the result from changes in rate of transcription, but mRNA stabilities. Using computational and experimental approaches, we identify a role of miR-145 in coregulating both Twist and Sox-9 to enhance N-cadherin due to p70S6K activation. We also provide evidence that this tumorigenic activity is associated with the ability of p70S6K to regulate miRNA biogenesis through tristetraprolin/Dicer interaction. To date, while the downstream targets of miRNAs are well defined, the underlying mechanisms of how miRNAs are deregulated are largely unknown. Our results shed new light on the p70S6K as a novel, important regulator of miRNA biogenesis that may underlie poor outcome in ovarian cancer. Journal article: #4 Conference proceedings: #2 [awarded ECCO Fellowship Grant], #3 Taken together, our data strongly support the important role for p70S6K in ovarian cancer metastasis and provide a mechanistic rationale for targeting p70S6K or its associated signaling in the treatment of metastatic ovarian cancer. Journal article: #1, #3 Book chapter: #6
Potential for further development of the research
and the proposed course of action:		 We reveal the novel identification of p70S6K regulation on both MCS formation and MCS-matrix adhesion that have pivotal roles in the metastatic progression of ovarian cancer. This reveals a new direction for understanding the oncogenic role of p70S6K in ovarian cancer metastasis and implicate that p70S6K could be a promising therapeutic target. Given the potential side effects of mTOR inhibitors, it is of great interest to develop a safe and effective therapeutic approach. We and others have demonstrated the use of siRNA to efficiently and specifically downregulate “undruggable” genes. However, one major limitation is targeted RNA delivery. We have developed vesicle-like nanostructures based on dendrimers as effective carriers for siRNA delivery in novel RNAi therapeutic applications (Kala et al., (2014) J. Med. Chem. 57: 2634-2642). We are actively pursuing in this direction. To expand our understanding of the complex mechanisms of action of p70S6K, we plan to take a proteomic approach to reveal a portrait of the entire signaling network. We will also examine p70S6K mediated bidirectional interactions between ovarian cancer cells and tumor microenvironment, leading to increased malignancy. These findings should highlight the functional importance of tumor-host crosstalk in impacting malignant cell behavior and implies important clinical implications.
Layman's Summary of
Completion Report:		 Ovarian cancer is highly lethal and the major form of cancer death of all gynecological malignancies. No effective therapy is currently available, with a poor 5-year survival rate (<25%). Thus, there is a need for new therapeutic targets and a better understanding of the molecular mechanisms underlying the progression of ovarian cancer. MCS mimic most of the in vivo properties and key hallmarks of cancer. Several of our new findings of this project indicate that p70S6K may contribute to the MCS formation and expansion and that it could be a promising target. (i) We show for the first time a new role for p70S6K on MCS formation through N-cadherin, modulating both adhesion and outside-in signaling. (ii) We also present evidence suggesting p70S6K in the tumor microenvironment, such as MCS-mesothelium communication, which is a rate-limiting step for the onset of the metastatic cascade. (iii) We identify p70S6K as a novel regulator of global miRNA biogenesis that may underlie the poor outcome in ovarian cancer. These results reveal a new direction for understanding the oncogenic roles of p70S6K and highlight its importance as a viable therapeutic target. Targeting p70S6K may thus be a useful approach in the treatment of ovarian cancer metastasis.
Research Output
Peer-reviewed journal publication(s)
arising directly from this research project :
(* denotes the corresponding author)
Year of
Publication		 Author(s) Title and Journal/Book Accessible from Institution Repository
2012 Ip CK, Wong AST*  Exploiting p70 S6 kinase as a target for ovarian cancer. Expert Opin Ther Targets 16: 619-630.  Yes 
2014 Ip CK, Yung S, Chan TM, Tsao SW, and Wong AST*  p70 S6 kinase drives ovarian cancer metastasis through multicellular spheroid-peritoneum interaction and P-cadherin/b1 integrin signaling activation. Oncotarget 5: 9133-9149.  Yes 
2014 Kala S, Mak AS, Liu X, Posocco P, Pricl S, Peng L, and Wong AST*  Combination of dendrimer-nanovector-mediated small interfering RNA delivery to target Akt with the clinical anticancer drug paclitaxel for effective and potent anticancer activity in treating ovarian cancer. J Med. Chem. 57: 2634-2642  Yes 
Lam SS, Ip CK, Mak AS, and Wong AST*  p70 S6 kinase is a regulator of microRNA biogenesis and influences the multicellular spheroid formation of ovarian cancer. Cancer Res. (submitted)  No 
Lam SS, Ip CK, and Wong AST*  p70 S6 kinase drives multicellular spheroid formation of ovarian cancer cells through a crosstalk mediated by canonical Wnt/b-catenin and non-canonical Wnt/Ca2+ pathways  No 
To SK, Lam SS, Tang KS, Li S, and Wong AST*  Cell adhesion in ovarian cancer. In Cancer Therapy: Recent Progress and Future Perspectives, Bentham Science Publishers (Book Chapter) [invited]  No 
Recognized international conference(s)
in which paper(s) related to this research
project was/were delivered :
Month/Year/City Title Conference Name
Sep 2013, Amsterdam, Netherlands p70 S6 kinase promotes a critical step in ovarian cancer metastasis by modulating tumor- mesothelial cell adhesion through a P-cadherin- b1-integrin crosstalk.  17thECCO-28thESMO-32ndESTRO European Cancer Congress 
Sep 2013, Amsterdam, Netherlands MicroRNA-145, a p70 S6 kinase-activated microRNA, regulates N-cadherin expression and epithelial- mesenchymal transition  17thECCO-28thESMO-32ndESTRO European Cancer Congress 
Apr 2014, San Diego, CA, USA p70 S6 kinase signals tristetraprolin/Dicer-mediated maturation of microRNA-145 to regulate tumor metastasis  American Association for Cancer Research Annual Meeting 
Other impact
(e.g. award of patents or prizes,
collaboration with other research institutions,
technology transfer, etc.):		 The PI would like to thank RGC for its support. In addition to publication in top-tier journal, there are also prizes and awards for research achievements and plenary/invited lectures that are direct outcomes from RGC support of this project: Prize and Awards for research achievements: 2012 Croucher Senior Research Fellowship (PI) 2013 17thECCO-28thESMO-32ndESTRO European Cancer Congress Fellowship Grant (Ip CK) 2013 17thECCO-28thESMO-32ndESTRO European Cancer Congress Fellowship Grant (Lam SS) Conference lecture / Invited Speaker: 1) Faculty of Health Science, University of Macau, Macau, 2015 Title: Cell adhesion in ovarian cancer: New concepts and prospects 2) Department of Life Sciences, Ewha Womans University, Seoul, Korea, 2014 Title: Cadherins in ovarian cancer: Complexity of a paradigm 3) College of Pharmacy, Kyung Hee University, Seoul, Korea, 2014 Title: Cadherins in ovarian cancer: Complexity of a paradigm 4) Kimmel Cancer Center, Philadelphia, PA, USA, 2013 Title: Cadherin switch and ovarian cancer 5) Renji Stem Cell Center/Shanghai Jiao Tong University, Shanghai, China, 2013 Title: Cadherins in ovarian cancer: paradigm or the odd one out 6) Croucher Advanced Study Institute: Tumor Microenvironment – New Concepts and Molecular Mechanisms Symposium, Hong Kong, 2012 Title: Cell-cell contacts in ovarian tumor metastasis 7) TEMTIA – V, Singapore, 2011 Title: p70 S6 kinase links EMT, metastasis, and the stem-like phenotype Collaboration: We have set up collaboration with Prof. J. Yates (Distinguished Visiting Professor, HKU & Scripps Research Institute, La Jolla, CA, USA) to identify the new targets of p70S6K in relation to this project. This is one of the two projects that Prof. Yates has identified from Biological Sciences, HKU which has good potential. The D3E-E389 and ΔN45ΔC105 constructs were kindly provided by Prof. G. Thomas (University of Cincinnati, OH, USA), who is the leader in this field.
  SCREEN ID: SCRRM00542