第一部分：胰腺癌被称为“癌中之王”，是最凶险的恶性肿瘤之一，患者五年生存率位于所有肿瘤的最末位。胰腺导管腺癌（PDAC）约占胰腺癌的90%。由于PDAC对免疫治疗和靶向治疗不敏感，手术切除获益有限，化疗仍是PDAC重要的治疗选择。吉西他滨是晚期/转移性PDAC、新辅助方案和切除后化疗中最常选择的药物，而吉西他滨耐药是影响PDAC化疗疗效的最大因素。因此，迫切需要阐明吉西他滨耐药的分子机理，寻找潜在治疗靶点，以期改善PDAC患者的治疗效果。值得注意的是，约90%的PDAC患者携带KRAS基因突变，该突变通过调控细胞氧化应激和代谢重编程等驱动肿瘤进展，也可能影响化疗敏感性。目前，研究发现靶向铁死亡是治疗PDAC以及逆转吉西他滨耐药的重要策略，而铁自噬的过度激活引起铁过载，是铁死亡启动的关键因素。

YB-1（Y-box binding protein 1）蛋白是一种多功能的分子，可分别与DNA、RNA以及蛋白互作，被发现在多种肿瘤中高表达且发挥促癌作用，在2019年被鉴定为RNA分子m5C修饰的识别蛋白。5-甲基胞嘧啶（5-methylcytosine，m5C）修饰是一种可逆的表观遗传修饰，参与调节多种肿瘤的发生发展、侵袭转移及治疗抵抗。本研究旨在探究YB-1是否通过识别m5C修饰、调控铁死亡途径在PDAC中发挥抵抗吉西他滨作用及其机制，以期找到新的治疗靶点。

本研究使用吉西他滨处理胰腺癌细胞，通过CCK8实验、平板克隆实验及xCELLigence细胞生长检测系统检测，结果显示YB-1高表达后，吉西他滨的IC50值升高、对于吉西他滨的敏感性降低。同时发现YB-1也可降低胰腺癌细胞对于泛KRAS抑制剂RMC-6236的敏感性。检测细胞内活性氧、亚铁离子以及脂质过氧化物水平，透射电镜下观察线粒体形态发现，YB-1蛋白过表达可抑制胰腺癌细胞的铁死亡；而检测细胞内自噬小体形成情况、铁自噬相关蛋白NCOA4与FTH1蛋白表达发现，YB-1可以抑制铁自噬过程。同时，通过分析胰腺癌耐药/敏感细胞系的差异基因，我们也发现YB-1是耐药组中高表达的基因，且差异基因的通路富集中可以显著富集到铁死亡相关通路。重要的是，抑制YB-1联合铁死亡诱导剂增加吉西他滨敏感性，下调FTH1的表达也可以逆转YB-1过表达引起的吉西他滨敏感性增高。接着，我们通过MeRIP-qPCR实验发现，具有降解NCOA4蛋白功能的E3泛素连接酶TRIM7存在m5C修饰的位点，并通过RIP实验验证YB-1作为TRIM7 m5C修饰的识别蛋白，且Western Blot实验显示YB-1过表达可促进TRIM7的蛋白水平升高。这提示我们YB-1通过结合TRIM7 m5C修饰位点，促进其蛋白水平升高，进而促进其对NCOA4的降解。利用YB-1冷休克结构域缺失的截短体和W65A点突变体质粒，我们发现YB-1通过发挥m5C识别功能介导胰腺癌铁死亡与细胞对于吉西他滨的敏感性。以上结果提示，YB-1可能通过TRIM7-NCOA4-FTH1轴调控铁死亡进而降低PDAC对吉西他滨的敏感性。

本研究聚焦于胰腺癌对于吉西他滨耐药这一临床难点，靶向铁死亡和m5C甲基化修饰这两大关键因素，深入探究m5C识别蛋白YB-1通过抑制铁自噬过程，降低胞内活性氧、亚铁离子以及脂质过氧化物水平，抑制铁死亡从而促进吉西他滨耐药的机制，为寻找潜在靶点在胰腺癌治疗的应用提供理论基础。

第二部分：

乳腺癌是全球范围内的健康问题，也是女性中发病率最高的恶性肿瘤之一。2022年，乳腺癌成为中国女性的第五大癌症死亡原因。尽管近年来乳腺癌的治疗方式不断优化，包括内分泌治疗、靶向治疗和免疫治疗等，但部分患者仍面临治疗耐药和疾病复发的问题，因此亟需探索其机制以开发更有效的治疗策略。

乳腺癌根据分子分型可分为多种亚型，其中ER⁺HER2^-型乳腺癌是最常见的亚型。核糖体蛋白S6激酶B1（S6K1）作为mTOR信号通路的重要下游分子，在乳腺癌中高频扩增，并与患者的耐药性和不良预后密切相关。S6K1在调控mRNA翻译中发挥重要作用，我们的前期研究通过Ribo-seq、RNA-seq及质谱技术系统性地解析了S6K1调控的全基因组翻译谱，并发现S6K1扩增可通过促进ER⁺HER2^-型乳腺癌细胞中CLU的翻译，增强自噬小体形成，从而促进自噬。然而，这一现象是否在其他乳腺癌亚型中普遍存在、S6K1如何通过CLU调控自噬过程以及S6K1的磷酸化是否在其中发挥作用等问题仍需进一步研究。

在本研究中，我们首先验证了S6K1在不同乳腺癌亚型中的蛋白表达水平，发现MCF7细胞中的S6K1蛋白表达显著高于其他细胞系。验证多组学分析确定的下游蛋白表达，发现我们前期对于S6K1的多组学检测和数据分析是高度可靠的。随后，在三阴性乳腺癌细胞系和HER2⁺乳腺癌细胞系中，我们发现S6K1同样通过上调CLU的翻译水平促进了自噬过程。免疫荧光实验显示，在正常、血清饥饿和巴佛洛霉素A1处理条件下，敲降S6K1显著减少了CLU与LC3的共定位。此外，我们利用雷帕霉素抑制S6K1的磷酸化，通过Western Blot检测发现，下游蛋白FYCO1、USP42、INCENP和CLU的翻译水平均受到抑制，进一步证实S6K1磷酸化对其翻译调控的作用。

本研究全面阐释了S6K1通过调控CLU翻译促进乳腺癌细胞自噬的分子机制，补充了不同乳腺癌亚型中这一过程的差异性，并揭示了S6K1磷酸化在其中的重要作用。这一发现不仅为乳腺癌的亚型特异性治疗提供了新思路，还为靶向自噬调控策略的开发奠定了理论基础。

Pancreatic cancer, known as the "king of cancers," is one of the most lethal malignancies, with the lowest five-year survival rate among all cancer types. Pancreatic ductal adenocarcinoma (PDAC) accounts for approximately 90% of pancreatic cancer cases. Due to its poor response to immunotherapy and targeted therapy, and the limited benefits of surgical resection, chemotherapy remains the primary treatment option for PDAC. Gemcitabine is the most commonly used drug for advanced and metastatic PDAC, or adjuvant chemotherapy after resection. However, gemcitabine resistance is the major factor compromising its efficacy in PDAC treatment. Thus, elucidating the molecular mechanisms underlying gemcitabine resistance and identifying potential therapeutic targets are urgently needed to improve treatment outcomes for PDAC patients. Notably, approximately 90% of PDAC patients harbor KRAS gene mutations, which drive tumor progression by regulating cellular oxidative stress and metabolic reprogramming, and may also influence chemotherapy sensitivity. Recent studies have shown that targeting ferroptosis is a promising strategy for treating PDAC and overcoming gemcitabine resistance, with excessive activation of ferritinophagy-induced iron overload being a key trigger of ferroptosis.

YB-1 (Y-box binding protein 1) is a multifunctional molecule that interacts with DNA, RNA, and proteins. It is overexpressed in various cancers, where it exerts oncogenic effects. In 2019, YB-1 was identified as an m⁵C (5-methylcytosine)-binding protein for RNA molecules. m⁵C is a reversible epigenetic modification involved in regulating tumorigenesis, progression, metastasis, and therapeutic resistance. This study aims to explore whether YB-1 mediates gemcitabine resistance in PDAC through m⁵C recognition and ferroptosis regulation, thereby identifying novel therapeutic targets.

In this study, pancreatic cancer cells were treated with gemcitabine, and cell viability was assessed using CCK-8 assay, colony formation assay, and xCELLigence real-time cell analysis system. The results showed that high YB-1 expression led to an increased IC50 of gemcitabine, indicating reduced drug sensitivity. Notably, YB-1 also reduced the sensitivity of pancreatic cancer cells to the pan-KRAS inhibitor RMC-6236. Ferroptosis markers, including reactive oxygen species (ROS), ferrous ion (Fe²⁺), and lipid peroxides (LPO), were significantly reduced, and mitochondrial morphology was preserved, as observed under transmission electron microscopy. Additionally, YB-1 suppressed ferritinophagy by decreasing NCOA4 protein levels and increasing FTH1 protein levels. Differential gene analysis between gemcitabine-resistant and sensitive PDAC cell lines revealed YB-1 as a highly expressed gene in resistant cells, with significant enrichment in ferroptosis-related pathways. Importantly, silencing FTH1 with siRNA reversed gemcitabine resistance induced by YB-1 overexpression. Mechanistically, m5C-modified sites were identified on TRIM7, an E3 ubiquitin ligase that degrades NCOA4, using MeRIP-qPCR. We found that YB-1 recognized the m5C modification on TRIM7 mRNA, as confirmed by RIP assays, and its overexpression increased TRIM7 protein levels, thereby promoting the degradation of NCOA4 Using YB-1 truncation constructs lacking the cold shock domain and the W65A point mutant plasmid, we found that YB-1 mediates ferroptosis and gemcitabine resistance in pancreatic cancer through its m5C recognition function. These findings suggest that YB-1 mediates gemcitabine resistance in PDAC through the TRIM7-NCOA4-FTH1 axis by inhibiting ferroptosis.

In summary, this study addresses the clinical challenge of gemcitabine resistance in PDAC, focusing on the key roles of ferroptosis and m5C methylation. It elucidates how YB-1 inhibits ferritinophagy, reduces intracellular ROS, Fe²⁺, and LPO levels, and suppresses ferroptosis to promote gemcitabine resistance. These findings provide a theoretical basis for exploring potential therapeutic targets for PDAC treatment.

2. 

Breast cancer is a global health issue and the most prevalent malignancy among women. In 2022, breast cancer was the fifth leading cause of cancer-related death in Chinese women. Despite advancements in breast cancer treatments, including endocrine therapy, targeted therapy, and immunotherapy, some patients still face challenges such as treatment resistance and disease recurrence. Therefore, it is crucial to explore the underlying mechanisms to develop more effective therapeutic strategies.

Breast cancer is classified into several molecular subtypes, with ER⁺HER2^- being the most common. Ribosomal protein S6 kinase B1 (S6K1), a critical downstream effector of the mTOR signaling pathway, is frequently amplified in breast cancer and is associated with therapy resistance and poor prognosis. S6K1 plays a vital role in mRNA translation regulation. Our previous studies systematically analyzed the genome-wide translational landscape regulated by S6K1 using Ribo-seq, RNA-seq, and proteomics. We demonstrated that S6K1 amplification promotes CLU translation, enhances autophagosome formation, and facilitates autophagy in ER⁺HER2^- breast cancer cells. However, it remains unclear whether this phenomenon occurs in other breast cancer subtypes, how S6K1 regulates autophagy through CLU, and whether S6K1 phosphorylation is involved in this process.

In this study, we first validated the protein expression levels of S6K1 in cell lines across different breast cancer subtypes. The results showed that S6K1 expression in MCF7 cells was significantly higher than in other cell lines. Validation of downstream proteins identified through multi-omics analyses confirmed the high reliability of our previous data. Furthermore, in triple-negative and HER2⁺ breast cancer cell lines, S6K1 was also found to promote autophagy by upregulating CLU translation. Immunofluorescence experiments revealed that S6K1 knockdown significantly reduced CLU and LC3 colocalization under normal, serum starvation, and bafilomycin A1 treatment conditions. Moreover, rapamycin-mediated inhibition of S6K1 phosphorylation suppressed the translation of downstream proteins FYCO1, USP42, INCENP, and CLU, as confirmed by Western blot analysis, further highlighting the critical role of S6K1 phosphorylation in translational regulation.

This study elucidates the molecular mechanism by which S6K1 regulates autophagy via CLU translation in breast cancer cells, highlights the differences across breast cancer subtypes, and underscores the importance of S6K1 phosphorylation in this process. These findings provide novel insights into subtype-specific therapies for breast cancer and lay a theoretical foundation for developing strategies targeting autophagy regulation.