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標題: | KRASG12C突變癌症之sotorasib抗藥性及藥物依存機制 Mechanisms of sotorasib resistance and addiction in KRASG12C-mutant cancer |
作者: | 邱俐雯 Li-Wen CHIOU |
指導教授: | 鄭永銘 Yung-Ming Jeng |
關鍵字: | sotorasib,藥物依存,KRAS,複製壓力,有絲分裂災難, sotorasib,drug addiction,KRAS,replication stress,mitotic catastrophe, |
出版年 : | 2023 |
學位: | 博士 |
摘要: | 美國食品及藥物委員會(US Food and Drug Administration)於2021年核可sotorasib(又名AMG510;商品名為Lumakras)用於治療帶有KRASG12C突變之局部晚期或轉移性非小細胞肺癌(non-small cell lung cancer),為首個被核准供臨床使用之KRASG12C抑制劑。儘管sotorasib在臨床試驗中達到優異的治療成效,這些帶有KRASG12C突變之癌細胞卻可能會在治療後產生抗藥性。大多數針對抗藥機制的研究表示,sotorasib抗藥性起因於癌細胞可藉由重新活化mitogen-activated protein kinase(MAPK)訊息路徑來躲避sotorasib對KRASG12C分子的抑制,因此瞭解參與抗藥性產生的訊息路徑及分子,有助於研擬更有效的治療方針及增進治療成效。
本篇研究中,我們自具有KRASG12C突變之胰臟癌及非小細胞肺癌細胞株建立出sotorasib抗藥性細胞株,並發現這些細胞會持續活化EGFR/PI3K/AKT訊息路徑;因此,若以PI3K抑制劑alpelisib或copanlisib合併sotorasib處理細胞,對於降低細胞存活率有加乘效果;反之,若過度表現持續性活化的PI3K 突變基因或是以shRNA降低PI3K的負調節分子PTEN的表現,則會促進細胞對sotorasib的抗藥能力。 另外,在我們研究抗藥性的過程中意外的發現到:以不含sotorasib的培養基培養抗藥性細胞時,這些細胞會表現出對sotorasib的依存性,因而降低生長速度並造成細胞死亡。在探討此依存性機制的結果中顯示:抗藥性細胞在剝奪sotorasib後會進行由p21Waf1/Cip1介導的生長停滯及由caspase介導的細胞死亡;高度活化的MAPK訊息路徑則會引起嚴重的DNA損傷及複製壓力(replication stress),造成DNA損傷反應(DNA damage response)的活化;持續性的活化MAPK訊息路徑及DNA損傷反應的耗盡會造成細胞過早進入有絲分裂,導致細胞分裂異常並造成微核(micronucleus)及核質橋(nucleoplasmic bridges)產生,進而發生有絲分裂災難(mitotic catastrophe)。若以第一型BRAF抑制物促進MAPK路徑過度活化,則能加劇抗藥性細胞在剝奪sotorasib後的死亡情形,並且在細胞實驗及動物實驗中皆得到證實。 總結而言,我們在本篇研究中證明了KRASG12C突變細胞會藉由活化未受sotorasib抑制的EGFR/PI3K/AKT訊息路徑獲得抗藥性,並闡明抗藥性細胞依存sotorasib的機制。高度活化的MAPK訊息路徑、DNA受損、複製壓力及有絲分裂災難是導致此類抗藥性細胞在剝奪sotorasib後細胞死亡的原因。此外,我們提供了以第一型BRAF抑制劑加劇此sotorasib依存現象的策略,期許為癌症患者提供更好的治療效果。 Sotorasib is the first KRASG12C inhibitor approved by the US Food and Drug Administration (FDA) for treating KRASG12C-mutant locally advanced or metastatic non-small-cell lung cancer (NSCLC) in 2021. Clinical trials on the therapeutic use of sotorasib for cancer treatment have reported promising results. However, KRASG12C-mutant cancers can acquire resistance to sotorasib after treatment. Most of the drug resistance mechanisms converge on reactivation of the mitogen-activated protein kinase (MAPK) pathway to bypass KRAS inhibition. Identifying the pathways and molecules involving in the resistance is critical for improving treatment response and developing more effective combination strategies. We established sotorasib-resistant (SR) cells using KRASG12C-mutant pancreatic cancer and NSCLC cell lines. Our data showed that the epidermal growth factor (EGFR)/phosphoinositide-3 kinase (PI3K)/AKT pathway was constitutively active in SR cells. Therefore, the PI3K inhibitor, alpelisib or copanlisib, in synergy with sotorasib reduced viability of both KRASG12C-mutant cancer cells and their SR sublines. On the contrary, overexpression of constitutively active PI3K mutant or shRNA-mediated knockdown of PTEN in KRASG12C-mutant cancer cells conferred sotorasib resistance. Moreover, during study on the mechanisms underlying sotorasib resistance, we incidentally discovered that when cultured in sotorasib-free medium, SR cells demonstrated a “drug addiction” phenomenon to decrease growth rate and increase cell death. We then investigated the mechanisms underlying sotorasib addiction. In the results, the sotorasib-resistant cells underwent p21Waf1/Cip1-mediated cell cycle arrest and caspase-dependent apoptosis in the absence of sotorasib. Sotorasib withdrawal resulted in robust activation of MAPK pathway, inducing severe DNA damage and replication stress, which activated the DNA damage response (DDR) pathway. Persistent MAPK pathway hyperactivation with DDR exhaustion led to premature mitotic entry and aberrant mitosis, followed by micronucleus and nucleoplasmic bridge formation, resulting in mitotic catastrophe. Pharmacologic activation of the MAPK pathway with a type I BRAF inhibitor could further enhance the effects of sotorasib withdrawal on sotorasib-resistant cancer cells both in vitro and in vivo. Collectively, we identified the EGFR/PI3K/AKT signaling as the bypass activated pathway that mediates sotorasib resistance and elucidated the mechanisms underlying the sotorasib addiction in KRASG12C-mutant cancer cells. Sotorasib addiction appears to be induced by hyperactivation of MAPK pathway, DNA damage, replication stress, and mitotic catastrophe. Furthermore, we devised a therapeutic strategy involving a type I BRAF inhibitor to strengthen the effects of sotorasib addiction; this strategy may provide clinical benefit for patients with cancer. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/89618 |
DOI: | 10.6342/NTU202302275 |
全文授權: | 同意授權(全球公開) |
顯示於系所單位: | 病理學科所 |
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