DESCRIPTION OF CURRENT RESEARCH
We study molecular mechanisms of intracellular membrane trafficking and signaling in health and disease. We seek to understand how endosomal compartments contribute to the trafficking and signaling of receptors for growth factors and cytokines and how the dysfunction of endosomes affects cell physiology. We are particularly interested in alterations that occur in signaling and trafficking processes in cancer cells because such changes may represent vulnerabilities of cancer cells to specific therapies. In parallel, we are also interested in trafficking pathways that operate in specific cell types or certain stages of cell differentiation.
In one of our previous projects, we described inflammatory signaling that was induced intracellularly upon endosome dysfunction (Mamińska et al., Sci Signal, 2016). As an underlying molecular mechanism, we found that the aberrant endocytic trafficking of cytokine receptors can cause their accumulation on endosomal membranes and the ligand-independent activation of nuclear factor-κB signaling, resulting in a fully engaged inflammatory cellular response. This mechanism occurs upon the dysfunction of components of endosomal sorting complexes required for transport (ESCRT) and is evolutionarily conserved from fly to human cells.
In our molecular oncology projects, we discovered synthetic lethality between two paralogous ATPases of ESCRT machinery, VPS4A and VPS4B (Szymańska et al., EMBO Mol Med, 2020). We showed that the VPS4B gene was frequently deleted in many cancer types, including in colorectal cancer, reflected by low VPS4B mRNA and protein levels in colorectal cancer samples from patients. The perturbation of VPS4A protein in tumor cells with the loss or low levels of VPS4B induced the death of cells that were grown in vitro and in a tumor xenograft model in mice. Moreover, upon the concomitant depletion of VPS4A and VPS4B proteins, dying cancer cells secreted immunomodulatory molecules that mediated inflammatory and anti-tumor responses. Overall, our results identified a novel pair of druggable targets for personalized oncology, thereby providing a rationale for developing VPS4 inhibitors for the precision treatment of VPS4B-deficient cancers. We also discovered lower gene expression of the ESCRT-I components VPS37A and VPS37B in colorectal cancer (Kolmus et al., J Cell Sci, 2021). At the molecular level, we showed that the concurrent depletion of VPS37 proteins evoked destabilization of the ESCRT-I complex and profound cellular stress responses.
Most recently, we revealed that the ESCRT-I complex is also indispensable for the biogenesis and functioning of lysosomes (Wróbel, Cendrowski et al., Life Sci Alliance, 2022). These organelles degrade various types of macromolecules that derive from endocytic and autophagic processes that ensure nutrient supply to fuel cellular metabolism. Lysosomes have lately gained much attention because targeting their function emerges as a promising strategy to treat cancer. We uncovered that the lack of ESCRT-I led to lysosome enlargement through inhibition of the degradation of their resident membrane proteins. This effect was accompanied by impairments in the delivery of internalized cholesterol to lysosomes. Using an RNA sequencing approach, we discovered that cells that lacked ESCRT-I activated transcriptional responses to counteract the improper delivery of nutrients that derive from lysosomal degradation. These responses involved the higher expression of genes whose products are known to induce the biosynthesis of cholesterol and de novo generation of lysosomes. We further revealed that these transcriptional changes resulted from the activation of TFEB/TFE3 transcription factors that are master regulators of autophagy and lysosome biogenesis. These factors can be activated by multiple cues. Upon ESCRT-I dysfunction, the activity of TFEB/TFE3 transcription factors was specifically induced by inhibition of the Rag GTPase-mTORC1 pathway. Overall, our results identify the ESCRT-I complex as an important regulator of lysosomal homeostasis (Fig. 1). Its function ensures the proper delivery of macromolecular cargo for degradation in lysosomes. This cargo is delivered from endosomes, autophagosomes, and lysosomal membranes. Consequently, ESCRT-I deficiency causes the improper supply of lysosome-derived nutrients, termed “lysosomal nutrient starvation”.
In another line of research, we focused on the receptor tyrosine kinase AXL, which is overexpressed in late-stage, metastatic, and drug-resistant cancers of various origins. Although the first AXL inhibitors are in clinical trials, cellular mechanisms of action of AXL remain unknown. By identifying the interactome of AXL, we revealed that the ligand-stimulated AXL receptor induces several actin-dependent processes (Zdżalik-Bielecka et al., Proc Natl Acad Sci U S A, 2021). Specifically, AXL activation induced the formation of circular dorsal ruffles and peripheral ruffles at the plasma membrane, macropinocytosis, and focal adhesion turnover. Such increases in membrane ruffling and macropinocytosis result in increases in the invasion and nutrient acquisition of cancer cells, respectively. We also characterized the endocytosis of AXL and discovered that ligand-bound receptors were rapidly internalized via several endocytic pathways, including both clathrinmediated and clathrin-independent routes (Poświata et al., Cell Mol Life Sci, 2022). The majority of the internalized receptor was not degraded but rather recycled back to the plasma membrane, coinciding with the sustained activation of AKT kinase signaling. Furthermore, we studied cellular effects of AXL inhibitors that are at various stages of clinical development (Zdżalik-Bielecka et al., Mol Cancer Res, 2022). We found that LDC1267 is a potent and specific inhibitor, whereas bemcentinib and gilteritinib exert off-target effects on cell growth and the endolysosomal and autophagy systems. These findings may help interpret results of ongoing clinical trials of AXL inhibitors.
Finally, while studying the cell type-specific regulation of membrane transport pathways during erythropoiesis, we identified cellular functions of a relatively poorly studied kinase, BMP2K, and its involvement in erythroid differentiation (Cendrowski et al., eLife, 2020; Cendrowski et al., Autophagy, 2020). We found that BMP2K acts in multiple membrane trafficking processes, including clathrin-mediated endocytosis, autophagy, and the regulation of COPII assemblies that are involved in secretion. Intriguingly, we found that two splicing variants of BMP2K (the longer BMP2K-L variant and shorter BMP2K-S variant) have partly different interactomes and exhibit opposite functions in SEC16A-dependent autophagy and erythroid differentiation. We propose that the BMP2KL/S regulatory system fine-tunes erythroid maturation through an unusual mechanism of two splicing ariants of a kinase that play opposing roles in intracellular processes.
