Impaired degradation of YAP1 and IL6ST by chaperone-mediated autophagy promotes proliferation and migration of normal and hepatocellular carcinoma cells.

Impaired degradation of the transcriptional coactivator YAP1 and IL6ST (interleukin 6 cytokine family signal transducer), two proteins deregulated in liver cancer, has been shown to promote tumor growth. Here, we demonstrate that YAP1 and IL6ST are novel substrates of chaperone-mediated autophagy (CMA) in human hepatocellular carcinoma (HCC) and hepatocyte cell lines. Knockdown of the lysosomal CMA receptor LAMP2A increases protein levels of YAP1 and IL6ST, without changes in mRNA expression. Additionally, both proteins show KFERQ-dependent binding to the CMA chaperone HSPA8 and accumulate into isolated lysosomes after stimulation of CMA by prolonged starvation. We further show that LAMP2A downregulation promotes the proliferation and migration in HCC cells and a human hepatocyte cell line, and that it does so in a YAP1- and IL6ST-dependent manner. Finally, LAMP2A expression is downregulated, and YAP1 and IL6ST expression is upregulated, in human HCC biopsies. Taken together, our work reveals a novel mechanism that controls the turnover of two cancer-relevant proteins and suggests a tumor suppressor function of CMA in the liver, advocating for the exploitation of CMA activity for diagnostic and therapeutic purposes.Abbreviations: ACTB: actin beta; ATG5: autophagy related 5; ATG7: autophagy related 7; CMA: chaperone-mediated autophagy; eMI: endosomal microautophagy; HCC: hepatocellular carcinoma; HSPA8: heat shock protein family A (Hsp70) member 8; IL6ST: interleukin 6 cytokine family signal transducer; JAK: Janus kinase; LAMP1: lysosomal associated membrane protein 1; LAMP2A: lysosomal associated membrane protein 2A; MAPK8: mitogen-activated protein kinase 8; P6: pyridine 6; SQSTM1: sequestosome 1; TUBA: tubulin alpha; VDAC1: voltage dependent anion channel 1; VP: verteporfin; YAP1: Yes1 associated transcriptional regulator.

RAF1 contributes to cell proliferation and STAT3 activation in colorectal cancer independently of microsatellite and KRAS status.

More than 30% of all human cancers are driven by RAS mutations and activating KRAS mutations are present in 40% of colorectal cancer (CRC) in the two main CRC subgroups, MSS (Microsatellite Stable) and MSI (Microsatellite Instable). Studies in RAS-driven tumors have shown essential roles of the RAS effectors RAF and specifically of RAF1, which can be dependent or independent of RAF's ability to activate the MEK/ERK module. In this study, we demonstrate that RAF1, but not its kinase activity, plays a crucial role in the proliferation of both MSI and MSS CRC cell line-derived spheroids and patient-derived organoids, and independently of KRAS mutation status. Moreover, we could define a RAF1 transcriptomic signature which includes genes that contribute to STAT3 activation, and could demonstrate that RAF1 ablation decreases STAT3 phosphorylation in all CRC spheroids tested. The genes involved in STAT3 activation as well as STAT3 targets promoting angiogenesis were also downregulated in human primary tumors expressing low levels of RAF1. These results indicate that RAF1 could be an attractive therapeutic target in both MSI and MSS CRC regardless of their KRAS status and support the development of selective RAF1 degraders rather than RAF1 inhibitors for clinical use in combination therapies.

An ERK-Dependent Feedback Mechanism Prevents Hematopoietic Stem Cell Exhaustion.

Hematopoietic stem cells (HSCs) sustain hematopoiesis throughout life. HSCs exit dormancy to restore hemostasis in response to stressful events, such as acute blood loss, and must return to a quiescent state to prevent their exhaustion and resulting bone marrow failure. HSC activation is driven in part through the phosphatidylinositol 3-kinase (PI3K)/AKT/mTORC1 signaling pathway, but less is known about the cell-intrinsic pathways that control HSC dormancy. Here, we delineate an ERK-dependent, rate-limiting feedback mechanism that controls HSC fitness and their re-entry into quiescence. We show that the MEK/ERK and PI3K pathways are synchronously activated in HSCs during emergency hematopoiesis and that feedback phosphorylation of MEK1 by activated ERK counterbalances AKT/mTORC1 activation. Genetic or chemical ablation of this feedback loop tilts the balance between HSC dormancy and activation, increasing differentiated cell output and accelerating HSC exhaustion. These results suggest that MEK inhibitors developed for cancer therapy may find additional utility in controlling HSC activation.