Colon cancer is one of the major cancers worldwide [1]. It is the fourth most common killer worldwide and the third most prevalent kind of cancer, with an estimated annual death toll of over 600,000 individuals due to colon cancer [2]. Patients with stage II or stage III colon cancer often have surgical resection, which is followed by adjuvant chemotherapy depending on clinical and pathological risk factors [3]. Initially, postoperative chemotherapy involved the use of fluoropyrimidine, but recently, combining fluoropyrimidine with oxaliplatin lowers risk of recurrence and increases survival rate of patients undergoing colon cancer resection. A recommended chemotherapeutic agent for the treatment of colon cancer is 5-Fluorouracil (5-FU) [4]. As an antimetabolite that inhibits cell proliferation, 5-FU mainly inhibits thymidine formation, which is essential for DNA synthesis, by blocking thymidylate synthase [5]. However, the development of acquired resistance in colon cancer cells greatly affects clinical application of 5-FU [6]. Overcoming chemoresistance is a challenge and gaining a deeper understanding of molecular mechanisms of 5-FU resistance regulation may open new avenues for colon cancer treatment.

In response to various stresses, cellular senescence, a very stable cell cycle arrest, occurs [7]. In tumor tissues, senescence is a collection of degenerative activities, including growth arrest, apoptosis resistance, chromatin remodeling, metabolic reprogramming, and phenotypic changes 8, 9, 10. The plasticity and reprogramming capability of senescent cells are considered crucial for emerging of resistant clones in numerous cancer types 11, 12, 13. Chemoresistance can arise through various mechanisms, including the production of therapy-induced senescence (TIS), a temporary growth arrest condition. In cancer, particularly in TIS, senescent cells themselves and the senescence-associated secretory phenotype (SASP) promote cell reprogramming into a stem cell state, leading to the emergence of resistance [14]. Studies have reported that regorafenib fosters resistance by making colorectal cancer cells senescence and epithelial-mesenchymal transition [15]. But little is understood about the molecular mechanisms underlying senescence involvement in colon cancer chemoresistance. This work aimed to explore the impact of TIS in colon cancer cells on 5-FU resistance.

INHBA (inhibin A) is a glycoprotein hormone secreted by the gonads, composed of α and βA subunits [16]. INHBA was previously thought to inhibit pituitary follicle stimulating hormone (FSH) synthesis and secretion [17]. INHBA has been associated with various cancers. INHBA overexpression is implicated in dismal prognosis in colon cancer and esophageal squamous cell carcinoma, and its expression is significantly linked to TNM staging 18, 19. INHBA promotes the malignant progression of colon cancer cells by upregulating VCAN [20], while metformin targets INHBA to suppress TGF-β/PI3K/AKT signaling and suppress the growth of colorectal cancer [21]. Furthermore, previous studies have indicated that INHBA is a novel mediator functioning as a senescence-associated modulator in colon cancer cells [22]. Importantly, INHBA is linked to chemoresistance in cancer cells [23]. Through bioinformatics analysis, we found that INHBA expression was upregulated in colon cancer, and patients with high INHBA expression showed poor prognosis. Furthermore, INHBA showed a significant positive correlation with senescence markers. But mechanism of INHBA in chemoresistance mediated by senescence in colon cancer cells remains unclear, making it crucial to dissect potential role of INHBA in regulating colon cancer chemoresistance.

Organ development and homeostasis depend heavily on the Hippo system, a signaling that controls cell proliferation, differentiation, and survival. Consequently, abnormal Hippo pathway function can result in a number of illnesses, including cancer [24]. The central elements of the Hippo pathway are MST1/2, SAV1, LATS1/2, YAP, and TAZ kinase cascade 25, 26, 27. When the Hippo pathway is active, LATS1/2-mediated phosphorylation represses action of YAP/TAZ. Dephosphorylation of YAP/TAZ by an inactive Hippo pathway leads in their translocation into the nucleus and subsequent interaction with the transcription factor TEAD1-4 to activate gene expression [28]. The inactivation of the Hippo pathway is considered a key mediator in the development of cancer chemoresistance. It has been reported that the decreased myosin phosphatase target subunit 1 (MYPT1) elevates ovarian cancer resistance to chemotherapy via repression of Hippo pathway signaling and induction of stemness [29], while transcription factor activating protein 2 γ (TFAP2C), through Hippo pathway inactivation, promotes stemness and chemoresistance in colon cancer [30]. Therefore, Understanding the processes behind Hippo pathway inactivation may offer fresh perspectives on overcoming chemoresistance in colon cancer.

Considering that INHBA may play a unique role in cell senescence and chemotherapy resistance, this work was to explore mechanism of INHBA in chemoresistance of colon cancer in vitro and in vivo by knocking down or overexpressing INHBA and whether to add inhibitor treatment. We manifested that INHBA was upregulated in colon cancer and that its knockdown could slow down cellular senescence and repress 5-FU resistance of colon cancer cells. Additionally, INHBA could promote cellular senescence and, thus, enhance chemoresistance in colon cancer cells via negative modulation of Hippo signaling. Our research provided a new perspective on mechanisms of 5-FU resistance in colon cancer cells and offered a strong basis for potential therapeutic strategies targeting senescent colon cancer.