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| Project Details |
| Funding Scheme : | General Research Fund | ||||||||||||||||
| Project Number : | 17103417 | ||||||||||||||||
| Project Title(English) : | Nuclear p70 S6 kinase: a molecular determinant of early tumor metastasis? | ||||||||||||||||
| Project Title(Chinese) : | 核p70S6激酶（p70S6K）：腫瘤轉移早期的分子性決定因子？ | ||||||||||||||||
| 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) : |
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| Panel : | Biology & Medicine | ||||||||||||||||
| Subject Area : | Biological Sciences | ||||||||||||||||
| Exercise Year : | 2017 / 18 | ||||||||||||||||
| Fund Approved : | 905,046 | ||||||||||||||||
| Project Status : | Completed | ||||||||||||||||
| Completion Date : | 30-6-2021 | ||||||||||||||||
| Project Objectives : |
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| Abstract as per original application (English/Chinese): |
目前，90%的癌症相關死亡可以歸因於癌轉移，因此了解癌轉移背後的分子機制顯得尤為重要。上皮間充質轉化（EMT）作為其機制之一，指的是上皮細胞失去胞間連接，並向間質表型轉化，從而獲得較高浸潤性和遊走遷移能力的現象。 EMT的發生是腫瘤轉移早期至關重要的一步，另外它還參與誘導腫瘤細胞的干性、耐藥性和免疫逃避性狀的發生。然而，目前對EMT的研究主要集中在其下游轉錄因子，EMT上游調節機制的研究尚不充分。 p70S6K是PI3K/AKT信號通路的下游效應因子，由於在人類腫瘤中常發現其存在活化突變，因此成為一個潛在的治療靶點。最近，我們第一次發現，p70S6K除了有已被充分證明的促腫瘤生存生長作用外，也參與調控了腫瘤發展的其他環節，如腫瘤轉移。而且，我們進一步發現p70S6K是通過誘導EMT的發生促進腫瘤轉移。需要強調的是，儘管p70S6K通常被當作一個細胞質蛋白，我們卻發現，p70S6K的腫瘤轉移調控功能與它的核定位相關。而異生胞核核糖核蛋白hnRNP A2/B2 （一個剪接體的重要組成部分）很有可能是p70S6K在細胞核中的作用蛋白，值得注意的是，我們的基因組分析也得到了一致的結果。目前已有證據顯示在一些正常細胞系中有核p70S6K的表達，但在癌組織中是否有核p70S6K的表達仍然缺少直接證據，而且，其入核機制也未見任何報導。在此，我們提出一種理論假設，即p70S6K通過在細胞核中調控mRNA前體的剪接產生不同的致癌變異體，誘導腫瘤細胞EMT的發生，進而促進腫瘤轉移。 通過體外體內方法，我們將（1）闡明hnRNPA2/B1蛋白的功能和它在核p70S6K表達腫瘤細胞中的促轉移機制； （2）澄清p70S6K的核轉移機制；（3）揭示hnRNPA2/B1在核p70S6K誘導的EMT中發揮在mRNA前體剪接方面的作用。 此研究不僅可以揭示腫瘤轉移前期的重要動態調控機制，也將會為腫瘤靶向治療的發展再添新的科學理論基礎。 |
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| Realisation of objectives: | Objective 1: To date, there is no study assessing the direct expression and function of p70S6K in the nucleus. We showed that nuclear localization of p70S6K increases with ovarian metastatic progression in human ovarian tumor samples and ovarian cancer cell lines, suggestive of its involvement in the regulation of nuclear functions. We showed that p70S6K is indeed a nucleo-cytoplasmic shuttling protein, and the nuclear import of p70S6K is required for efficient epithelial-to-mesenchymal transition (EMT). We further identified hnRNPA2/B1 as a novel nuclear p70S6K interaction partner. hnRNPA2/B1 siRNA, but not with nonspecific siRNA, exhibited a discernible reduction in the mesenchymal phenotype, with a concomitant decrease in N-cadherin/vimentin expression and an increase in E-cadherin expression. These data demonstrate an essential role of hnRNPA2/B1 in the regulation of EMT downstream of nuclear p70S6K Tumor xenografts in mice and patients’ clinical samples corroborate these findings in vivo. Percentage achieved: 100% Objective 2: Whether or not p70S6K needs to be phosphorylated for entering the nucleus is controversial. Our findings confirm an involvement of phosphorylation in regulating p70S6K nuclear localization upon stimulation of cytokines/growth factors that are highly expressed in ascitic fluid of ovarian cancer patients, such as hepatocyte growth factor. Interestingly, the kinase activity of p70S6K is indispensable for its nuclear translocation. First, the kinase-inactive p70S6K also underwent nuclear translocation. Second, overexpression of constitutively active mutant of p70S6K enhanced nuclear localization. Importin a1, a3 and a4, but not a2, and b1 could interact with p70S6K. Silencing Ran with siRNA caused a significant reduction of nuclear p70S6K. These data together demonstrate the active nucleocytoplasmic shuttling of p70S6K depending on exportin-1, importin, and Ran, rather than the presence of two static pools of p70S6K separated in the cytoplasm and the nucleus. Percentage achieved: 100% Objective 3: Pre-mRNA splicing is important in generating oncogenic variants with cancer-specific functions. p70S6K can be alternatively spliced to h6A and h6C isoforms. We showed that h6A and h6C were potent inducers of EMT, and h6A and h6C generation is a pre-requisite of p70S6K-mediated EMT. Moreover, with the Iso-seq, a significant increase in the splice variants of CASP9, BIN1, and RON, which have been previously implicated in the progression and metastasis of ovarian cancer, as well as new spliced variants were observed. Despite the absence of a consensus p70S6K phosphorylation motif, a non-consensus phosphorylation site (serine residue at position 73/85 (A2/B1) is on hnRNPA2/B1. Percentage achieved: 100% These findings not only reveal a previously unrecognized role for p70S6K in the nucleus, but also unveil a novel role for nuclear p70S6K in mediating the metastatic cascade through alternative splicing, which has been recognized as the prime driving event in almost all hallmarks of cancer. Nuclear p70S6K and its signaling molecules may represent more precise targets with potential therapeutic advantage for metastatic ovarian cancer. Publications (*, corresponding author) (Note: ALL as corresponding author): Journal article: 1. To SK, Zhou J, Ip PP, Chan KK, Lam SS, Fung YE, Che CM, and Wong AS* Nuclear p70 S6 kinase drives aberrant alternative splicing through hnRNP2A/B1 to mediate epithelial-to-mesenchymal transition and tumor progression. Nat. Cell Biol. (under review). 2. Artemenko M, Zhong SS, To SK, and Wong AS* (2022) p70 S6 kinase as a therapeutic target in cancers: more than just an mTOR effector. Cancer Lett. 535: 215593. [invited] (impact factor: 8.679). 3. Yi C, Lai SK, Tsang CM, Artemenko M, Tang MK, Pang SW, Lo KW, Tsao SW, and Wong AS* (2021) A 3D spheroid-specific role of Wnt/b-catenin and Eph/Ephrin in nasopharyngeal carcinoma cells. J. Cell Sci. 134: jcs256461. (impact factor: 4.573). Conference proceedings: 4. To SK, Zhou J, Wong AS*. p70 S6 kinase: a nuclear role in alternative splicing through hnRNPA2/B1 to mediate ovarian cancer metastasis. Metastasis Research Society 18th Biennial Congress, Virtual Meeting (Abstract No. 79) (2021). 5. Zhou J, To SK, and Wong AS*. Nuclear localization of p70 S6 kinase in ovarian cancer. AACR Annual Meeting 2018, Chicago, IL (Abstract No. 18-A-4365-AACR) (2018). Patent: 6. Utilization of nuclear p70 S6 kinase for the diagnosis, prognosis and treatment of cancer. Inventors: Wong AST et al. (1st inventor) (US Provisional Application No. 63/005,507) (Patent Cooperation Treaty) | ||||||||||||||||
| Summary of objectives addressed: |
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| Research Outcome | |||||||||||||||||
| Major findings and research outcome: | Objective 1: Here, using cancerous tissues and isogenic ovarian cancer cell models with opposite metastatic abilities, we provide the first evidence the presence of nuclear p70S6K, and also indicate a positive association between nuclear p70S6K level and metastatic tendency. Nuclear p70S6K promotes epithelial-to-mesenchymal (EMT) phenotypes and cell migration, and enhances peritoneal metastasis in mice, which is uncoupled from the growth stimulating function of cytoplasmic p70S6K. We further identify hnRNPA2/B1, a critical spliceosome component, as a novel nuclear p70S6K-interacting protein in EMT and metastasis. Publication: Journal article: #1 Conference proceeding: #4, 5 Objective 2: p70S6K is indeed a nucleo-cytoplasmic shuttling protein. On exploring how the p70S6K translocates into the nucleus, we show that the nuclear localization of p70S6K is phosphorylation-dependent, whereas its kinase activity is not necessary. Furthermore, we uncovered a potential nuclear localization signal and the active nucleocytoplasmic shuttling of p70S6K depends on exportin-1, importin a1, a3, a4 and b1 and the Ran GTPase, rather than the presence of two static pools of p70S6K separated in the cytoplasm and the nucleus. Publication: Journal article: #1 Conference proceeding: #4, 5 Objective 3: To explore the molecular mechanisms of nuclear p70S6K in promoting EMT, we have identified hnRNPA2/B1. We have demonstrated a critical role of hnRNPA2/B1 in the nuclear p70S6K-mediated EMT via modulating alternative splicing of RPS6KB1 pre-mRNA, h6a and h6c. With Iso-seq, a significant increase in the splice variants of CASP9, BIN1, and RON, which have been previously implicated in the progression and metastasis of ovarian cancer, as well as new spliced variants were observed. Despite the absence of a consensus p70S6K phosphorylation motif, a non-consensus phosphorylation site is on hnRNPA2/B1. Tumor xenografts mice and detection of both nuclear p70S6K/hnRNPA2/B1 and p70S6K short isoforms in patient samples indicate therapeutic potential of targeting these signaling molecules in vivo. Publication: Journal article: #1 Conference proceedings: #4, 5 Taken together, our findings not only shed new light on a new direction for understanding the oncogenic roles of p70S6K in ovarian cancer, but may also be extended to other cancers with inappropriate activation of p70S6K. Selective targeting of nuclear p70S6K activity or its downstream signaling represents a promising strategy to specifically target metastatic cells, and at the same time minimize the adverse side effects caused by mTOR inhibitors (e.g. rapamycin) that downregulate cytoplasmic p70S6K. Publication: Journal article: #1-3 Conference proceedings: #4, 5 Patent: #6 | ||||||||||||||||
| Potential for further development of the research and the proposed course of action: |
We reveal the novel identification of a nuclear role for p70S6K in both alternative splicing and EMT 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 (Ma et al., 2018, Mol. Ther. 26: 70-83). We are actively pursuing in this direction. The repurposing of approved or investigational drugs for other non-oncology diseases (e.g. metabolic diseases) for cancer therapy is emerging as a very effective strategy for drug discovery as it is with reduced time, cost and risk. In our pilot study, we show that riluzole, a FDA-approved drug for amyotrophic lateral sclerosis, could effectively suppress the nuclear p70S6K/hnRNPA2/B1-mediated EMT phenotype. We will continue to unravel the underlying mechanisms of action and explore its development with rationally designed drug combinations. Active p70S6K has also been detected in patients with breast, gastric, and colon cancer. To expand our understanding of the nuclear p70S6K, we plan to examine if this nuclear translocation is also seen in other tumor types in which active p70S6K is an important pathological process. These findings should highlight the functional importance of nuclear p70S6K in impacting aggressive malignant cell behavior and implies important clinical implications. | ||||||||||||||||
| Layman's Summary of Completion Report: | One of the greatest unmet needs compromising the successful treatment of cancer is metastasis. 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. Epithelial-to-mesenchymal transition (EMT) is an early key step in metastasis and that it could be a promising therapeutic target. (i) We show for the first time a new nuclear location of p70S6K, despite it is generally considered to be a cytoplasmic protein. (ii) We also present evidence suggesting this nuclear translocation of p70S6K in the EMT. (iii) We identify hnRNPA2/B1, a critical splicesome component, is a specific and novel p70S6K-interacting protein in the nucleus which generates distinct oncogenic variants that may underlie the EMT, metastasis, and 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 nuclear activity 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) |
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| Recognized international conference(s) in which paper(s) related to this research project was/were delivered : |
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| Other impact (e.g. award of patents or prizes, collaboration with other research institutions, technology transfer, etc.): |
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| SCREEN ID: SCRRM00542 |