Unlocking the metastatic switch: The RHEB-CSF1R complex ignites epithelial-mesenchymal transition in pancreatic cancer by autophagy

Abstract

This study, for the first time, unveils a novel mechanism in which the autophagy-related gene Ras homolog enriched in brain (RHEB) forms a complex with colony-stimulating factor 1 receptor, thereby modulating the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin signaling axis. This interaction suppresses autophagy and promotes epithelial-to-mesenchymal transition, ultimately driving pancreatic cancer metastasis. Beyond elucidating RHEB’s role as an oncogene and a predictor of poor prognosis, this research provides a theoretical foundation for developing innovative therapeutic strategies targeting the RHEB-colony-stimulating factor 1 receptor axis.

Key Words: Pancreatic cancer; Autophagy; Epithelial-to-mesenchymal transition; Colony-stimulating factor 1 receptor; Mammalian target of rapamycin; Ras homolog enriched in brain; Growth factors

Core Tip: Pancreatic cancer is among the malignancies with the poorest outcome. The lethal nature of this disease stems not from primary tumor growth, often resectable in the minority of patients presenting with localized disease, but from early, occult dissemination to distant organs. Consequently, understanding and targeting the molecular machinery driving metastasis represents the paramount challenge in pancreatic cancer therapeutics. In a recent study identified a novel protein complex between Ras homolog enriched in brain and colony-stimulating factor 1 receptor that regulates autophagy and epithelial-mesenchymal transition to promote pancreatic cancer metastasis. This article critically examines the significance of these findings, contextualizes them within the current understanding of mammalian target of rapamycin biology and tumor microenvironment interactions, and proposes the direction of mechanism research and therapeutic transformation.

INTRODUCTION

Mammalian target of rapamycin (mTOR) stands for the mammalian target of rapamycin, which regulates cellular signaling pathways and is a serine/threonine kinase[1-3], energy metabolism pathways[4], and certain amino acid metabolism pathways[5], regulating processes such as cell growth, proliferation, metabolism, and autophagy. mTOR complex 1 (mTORC1) is fully activated on the lysosomal membrane through a four-step cascade process[6-8]. First, mTORC1 is recruited to within approximately 100 angstroms of the lysosomal membrane by late endosomal/Lysosomal adaptor and mitogen-activated protein kinase and mTOR activator 1 anchoring of RAG-Ragulator complex. Second, the membrane-bound Ras homolog enriched in brain (RHEB)-guanosine triphosphate (GTP) further pulls mTORC1 closer to about 40 angstroms. Third, the Raptor subunit of mTORC1 contacts the membrane through its “FM finger” domain. Finally, a basic loop on the N-HEAT domain of the mTOR subunit binds to the membrane, driving the conformational change of mTORC1 to a fully active state. Furthermore, studies have found that the RAG-Ragulator complex, in addition to binding to Raptor, can also directly interact with the mammalian lethal with SEC13 protein 8 subunit of mTORC1 through Ras-related GTPase A-GTP, forming an additional membrane anchoring site. These interactions collectively stabilize the conformation of mTORC1 on the membrane and synergistically enhance the kinase activity of mTORC1 by more than 35-fold through RHEB-GTP and the RAG-Ragulator complex, thus achieving logical integration of growth factor signaling (via RHEB) and nutrient signaling (via RAGs) on the lysosomal membrane. The Raptor subunit of mTORC1 binds to the target of rapamycin signaling motifs of classic substrates such as S6K and 4E-BP1, phosphorylating these substrates, which then translocate to the nucleus to promote cellular anabolic metabolism. Activated mTORC1 inhibits autophagy by phosphorylating Unc-51 like kinase 1 (ULK1) and autophagy-related protein 13 (ATG13)[9-13]. transcription factor EB (TFEB) interacts directly with activated Rag GTPases (particularly GDP-bound RagC/D) through its N-terminal region, thereby being recruited near the mTORC1 complex for phosphorylation. Phosphorylated TFEB forms a complex with 14-3-3, leading to TFEB retention in the cytoplasm and inhibiting autophagy[5]. During nutrient deprivation, mTORC1 is inactivated, allowing ULK1, ATG13, and TFEB to be rapidly dephosphorylated, thus activating ULK1 kinase and inducing autophagy[9]. TFEB can then enter the nucleus to initiate the expression of genes related to lysosome biogenesis and autophagy[14].

RHEB is a highly conserved small GTPase evolutionarily, belonging to the Ras superfamily[15-17]. Its protein structure contains a typical GTP-binding domain and is anchored to intracellular membrane structures such as the lysosomal membrane through farnesylation modification at the C-terminus CaaX motif[18-20]. The core function of RHEB is to act as a key upstream activator of mTORC1. In the GTP-bound state, RHEB directly binds to and activates the kinase activity of mTORC1[6,21]. Tuberous sclerosis complex subunit 2 (TSC2) catalyzes the hydrolysis of GTP-bound RHEB, converting it to the inactive GDP-bound state[22-24], inhibiting mTORC1 activation and promoting autophagy. Growth factors such as insulin activate protein kinase B (AKT) through class I phosphatidylinositol 3-kinase (PI3K)-dependent pathways, and AKT phosphorylates and inactivates TSC2, relieving TSC2’s inhibition on RHEB, thereby activating mTORC1[25,26].

Colony-stimulating factor 1 (CSF1) receptor (CSF1R) is a tyrosine kinase receptor activated by CSF1 and interleukin-34, which can cause changes in a series of signaling pathways upon activation[27-29]. It can transform macrophages into M2 macrophages[30-32], mediating malignant progression such as tumor cell proliferation and invasion[27,33]. However, investigations into RHEB/mTOR’s involvement in pancreatic cancer remain limited, Whether there is a synergistic mechanism between RHEB and CSF1R is unknown, particularly concerning its contribution to the metastatic processes leading to malignancy. Therefore, clarifying the precise mechanism through which the autophagy-related gene RHEB/mTOR and CSF1R promote pancreatic cancer metastasis has the potential for improving prognosis as well as for developing new prevention and treatment strategies.

Pancreatic cancer has a poor outcome. Survival has been improving, but at a slower rate than other cancers[34-36]. Although research continues to deepen, there are still many limitations in the current research of pancreatic cancer, such as unclear metastasis mechanism, frequent drug resistance to targeted therapy, poor prognosis of patients and other core issues, which require further study.

The study by Deng et al[37] provides a new regulatory mechanism for tumor autophagy inhibition and epithelial-mesenchymal transition (EMT) progression. The RHEB-CSF1R complex promotes overactivation of the PI3K/AKT/mTOR signaling pathway, thus inhibiting autophagy, promoting the expression of EMT markers, and ultimately driving the migration and invasion of tumor cells. This study not only fills the gap in the mechanism of how autophagy and EMT are coordinated and regulated by a specific molecular complex (RHEB-CSF1R) in pancreatic cancer, but also has the potential to evaluate the prognosis of patients and develop new targeted therapies.

THE “NEW KEY” DISCOVERED: THE RHEB-CSF1R PROTEIN COMPLEX IS IDENTIFIED FOR THE FIRST TIME AS A PROMOTOR OF THE METASTATIC AXIS

CSF1R is central to cellular interactions within the TME, particularly in the development and function of macrophages[27,28]. In many tumors, the uncontrolled expression of CSF1R may be accompanied by the progression of TME. Additionally, CSF1R is highly expressed on the cell membrane of many tumors and is closely related to tumor proliferation, invasion, and other factors[28,29].

RHEB is highly expressed in non-small cell lung cancer[38], prostate cancer[2], some human lymphomas[39], hepatocellular carcinoma[40], pancreatic cancer[41], and other cancers[42]. RHEB is located on the surface of lysosomes[1,4-6], cytoplasm[43,44], endoplasmic reticulum and Golgi[45], mitochondrial membrane[46], and within the nucleus[18]. The abnormal subcellular localization of RHEB protein, particularly its excessive accumulation on the lysosomal membrane, is a key mechanism leading to the sustained activation of the mTORC1 signaling pathway and driving tumor growth. KRAS mutations were found to have a synergistic effect with transferase inhibitors in tumor models[47-51]. In tumor cells, it is still unknown whether RHEB has a synergistic effect with CSF1R.

Deng et al[37] through RNA-sequencing combined with co-immunoprecipitation, first confirmed that the small GTPase RHEB can directly bind to the tyrosine kinase receptor CSF1R to form a stable complex. This complex is highly expressed in primary pancreatic cancer lesions and liver/Lymph node metastatic lesions, and its expression level is significantly negatively correlated with the overall survival of patients (TCGA cohort hazard ratio = 1.82, P < 0.001). Functionally, overexpression of RHEB or restoration of CSF1R expression can significantly restore the migration and invasion abilities of cells subjected to knockdown. Conversely, dual-target silencing almost completely blocks the formation of micrometastases in a mouse spleen-liver metastatic model. This finding breaks through the traditional understanding that “RHEB regulates growth only through mTORC1”, expanding its function for the first time to the “initiation of metastasis” stage. It also provides a new research direction for the future research of pancreatic cancer: Structural mapping and functional verification of the RHEB-CSF1R complex binding interface, pharmacological screening and drug development for the RHEB-CSF1R axis, and clinical verification of RHEB-CSF1R as a diagnostic or prognostic biomarker.

POSITIVE FEEDBACK PATHWAY: RHEB-CSF1R ENHANCES THE ACTIVATION OF THE PI3K/AKT/MTOR PATHWAY

In the TME, tumor-associated macrophages secrete CSF1 factors to promote tumor cell proliferation and migration[28,52-56]. CSF1 binds to the cell membrane receptor CSF1R, activating cytoplasmic PI3K/AKT phosphorylation[27,57]. Phosphorylated AKT relieves the inhibition of RHEB by TSC2[25,26], and activated RHEB binds to lysosome-localized mTORC1 and activates it via phosphorylation[6,21]. Activated mTORC1 strongly promotes the biosynthesis of macromolecules such as proteins, lipids, and nucleotides, providing a material basis for cell proliferation[58-62]. However, interestingly, to meet the enormous demand for biosynthetic precursors due to this high-speed synthetic metabolism, tumor cells simultaneously require a high level of autophagic flux to provide “building materials”[43]. This necessitates that tumors develop sophisticated regulatory mechanisms to partially decouple mTORC1 activity from autophagy suppression, achieving the coexistence of “synthesis” and “recycling”. No farnesylated RHEB can enter the nucleus and activate mTORC1 within the nucleus, which may be related to the regulation of specific gene transcription and is relatively independent of autophagy regulation on the lysosomal membrane[18].

Deng et al[37] confirmed through in vitro and in vivo experiments that the RHEB-CSF1R complex promotes the proliferation, migration, EMT, and inhibition of autophagic flux (significantly downregulated light chain 3α/β/Beclin-1/ATG5) in pancreatic cancer cells by continuously enhancing the phosphorylation levels of PI3K (Tyr524), AKT (Ser473), and mTOR (Ser2448). Silencing RHEB can inhibit the proliferation, invasion, migration, and EMT of pancreatic cancer cells; reduce the formation of subcutaneous tumors and liver metastases in mice; and increase the number of autophagosomes and autolysosomes as well as the protein levels of autophagy markers light chain 3α/β, Beclin 1, and ATG5. Conversely, overexpression of RHEB produces the opposite effects. The inhibitory effect of CSF1R silencing on cell behavior was slightly weaker than that observed for RHEB silencing. Rescue experiments confirmed that a PI3K activator or the autophagy inhibitor chloroquine could partially reverse the inhibitory effect of RHEB silencing on cell migration, invasion, and EMT. In summary, RHEB forms a complex with CSF1R, activating the PI3K/AKT/mTOR pathway, thereby inhibiting autophagy and promoting EMT, ultimately driving the metastatic process of pancreatic cancer.

Therefore, Deng et al[37] speculate that a RHEB-CSF1R positive feedback loop activates the PI3K/AKT/mTOR pathway in pancreatic cancer (Figure 1). When CSF1 or growth factor binds to the corresponding membrane receptors, they activate the tumor cell PI3K/AKT/RHEB/mTOR pathway, while RHEB promotes the expression of CSF1R in pancreatic cancer through unknown pathways. RHEB forms a complex with CSF1R and localizes on the cell and/or subcellular membrane, further enhancing activation of the PI3K/AKT/mTOR pathway, thereby maintaining high expression of RHEB. Previous studies have confirmed that targeting RHEB in combination for tumor treatment is feasible[48]. This mechanism may partially explain the physiological mechanism of high RHEB expression in pancreatic cancer and provide theoretical support for targeting RHEB-CSF1R in the treatment of pancreatic cancer.

Figure 1 Growth factors inhibit autophagy and promote anabolic metabolism by the Ras homolog enriched in brain-colony-stimulating factor 1 receptor axis. The figure was generated by the Gemini 3 Pro Image model (version: Gemini-3-pro-image-preview) and Nano Banana. It illustrates that growth factors inhibit autophagy and promote anabolic metabolism, both mediated by activation of the phosphatidylinositol 3-kinase/protein kinase B/Ras homolog enriched in brain (RHEB)/mammalian target of rapamycin pathway. Additionally, a positive feedback loop exists between RHEB and colony-stimulating factor 1 receptor that activates the phosphatidylinositol 3-kinase/protein kinase B/RHEB/mammalian target of rapamycin pathway in pancreatic cancer. RHEB: Ras homolog enriched in brain; CSF1R: Colony-stimulating factor 1 receptor; AKT: Protein kinase B; PI3K: Phosphatidylinositol 3-kinase; mTOR: Mammalian target of rapamycin; EMT: Epithelial-mesenchymal transition; LC3: Light chain 3.

Despite the innovative findings in the study by Deng et al[37], this work was not without limitations. Firstly, the subcellular localization of the RHEB-CSF1R complex in pancreatic cancer cells remains unclear. Secondly, experiments have verified changes in the phosphorylation levels of the PI3K/AKT/mTOR pathway and their effects on cell migration, invasion, and autophagy, but the potential synergistic effects of other signaling pathways have not yet been fully excluded. Finally, the results of in vitro experiments cannot fully simulate the complex effects of the TME on autophagy and metastasis in vivo. Future research needs to combine more advanced in vitro models (such as organoids and co-culture with stromal cells) and in vivo imaging technologies to dynamically monitor autophagy and metastasis in the TME.

CONCLUSION

The authors present evidence suggesting that the RHEB-CSF1R complex influences pancreatic cancer metastasis through the regulation of autophagy. Specifically, this complex activates the PI3K/AKT/mTOR pathway to inhibit autophagy, thereby promoting EMT and tumor metastasis. These findings suggest that RHEB may serve as a biomarker for predicting pancreatic cancer prognosis and developing new therapeutic strategies. For tumor biologists seeking therapeutic targets or drug developers dedicated to overcoming the immune microenvironment barrier of pancreatic cancer, this study provides valuable insights and is highly recommended for further consideration.