Unraveling the potential and complex interplay of endolysosomal proteins TRPML1, TPC2, and Rab7a

Abrahamian, Carla (2024): Unraveling the potential and complex interplay of endolysosomal proteins TRPML1, TPC2, and Rab7a: implications for cancer and neurodegenerative disorders. Dissertation, LMU München: Medizinische Fakultät

Abstract

The endolysosomal network comprises distinct interconnected membrane-bound organelles, namely early endosomes (EEs), recycling endosomes (REs), multivesicular bodies (MVBs), late endosomes (LEs), and lysosomes (LYs). Within eukaryotic cells, LYs serve as primary degradative centers, housing a variety of enzymes that function optimally at their acidic pH and are capable of degrading proteins, lipids, and carbohydrates. Lysosomal function and physiology are regulated by resident proteins and ion channels that facilitate ion movement across the endolysosomal membrane. The transient receptor potential mucolipin channel 1 (TRPML1) and two-pore channel 2 (TPC2) are chief cation channels found in LEs and LYs and share several characteristics. These channels are permeable to calcium (Ca2+) and sodium (Na+) and govern cargo trafficking, vesicle fusion, and membrane dynamics in the endolysosomal system. Additionally, both TRPML1 and TPC2 interact with the mammalian target of rapamycin complex 1 (mTORC1), are involved in lysosomal exocytosis and autophagy, and are activated by phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2). Dysfunctions or mutations in endolysosomal ion channels have been associated with various channelopathies, encompassing autoimmune disorders, fatty liver disease, neurodegenerative diseases, lung disease, metabolic disorders, and cancer. A prominent group of disorders directly related to lysosomal pathophysiology is known as lysosomal storage diseases (LSDs). Mucolipidosis type IV (MLIV), the autosomal recessive LSD, arises from mutations in the gene encoding TRPML1 (MCOLN1). Typically, MLIV manifests in childhood with neurodegenerative symptoms accompanied by visual and motor impairments. MLIV patients often exhibit an accumulation of lipid products and increased aggregates, such as p62/Sequestosome 1 (SQSTM1), within intracellular organelles. In Scotto Rosato et al., 2022, we aimed to rescue LSD phenotypes by activating TPC2 using the selective agonist TPC2-A1-P. We utilized different models, including induced pluripotent stem cell (iPSC)-derived neurons, patient fibroblasts, and in vivo MLIV mice. Interestingly, stimulation of MLIV cells with TPC2-A1-P reduced lipid and cholesterol accumulation, reversed the autophagy blockade, and restored cellular ultrastructure. Additionally, MLIV mice treated with TPC2-A1-P ameliorated central nervous system defects and exhibited improved motor performance compared to mice treated with DMSO vehicle control. Besides LSDs, a study has revealed an intriguing connection between TRPML1 and triple-negative breast cancer (TNBC). TNBC is known for its aggressive nature, characterized by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) amplification. TRPML1 showed an upregulated expression in TNBCs, and its silencing inhibited the growth of these cancer cells by regulating the activity of mTORC1. For this purpose, we extended our work to investigate the proposed role of TRPML1 in TNBC. In Rühl et al., 2021, we successfully developed the first isoform-selective and potent antagonist for TRPML1, namely the steroid 17β-estradiol methyl ether (EDME). To decipher its mechanism of action, we treated the MDA-MB-231 TNBC cell line with EDME and generated a CRISPR/Cas9 TRPML1 knockout (KO) within the same cell line. The results were compelling, showing that both TRPML1 KO and EDME treatment reduced cell migration and invasion compared to the control. The TRPML1 KO line provided further evidence of the on-target effects of EDME. These findings offer valuable insight into the potential of temporarily modulating TRPML1 activity to suppress the growth and migration of aggressive TNBC. Nevertheless, despite key similarities, TRPML1 and TPC2 also possess distinct properties. Notably, TPC2 can also be activated by the Ca2+-mobilizing second messenger nicotinic acid adenine dinucleotide phosphate (NAADP), rendering the ion selectivity of TPC2, a highly debatable topic. A previous study in our laboratory demonstrated the agonist-dependent activation of TPC2 using small molecule activators, TPC2-A1-P (used in Scotto Rosato et al., 2022) mimicking PI(3,5)P2 activation, and TPC2-A1-N mimicking NAADP activation. Intrigued by the agonist-mediated switch of TPC2 between non-selective selective Ca2+ and selective Na+ states, we further investigated the simultaneous application of these compounds on TPC2 behavior in Yuan et al., 2022. To explore this aim, we tested the co-application of TPC2-A1-P and TPC2-A1-N using Ca2+ imaging via genetically encoded Ca2+ indicator GCaMP6 and electrophysiology patch clamp recordings. Our preliminary investigations aimed at testing various cell lines for the expression of TPC2 (data published in Abrahamian et al., 2021, appendix). The melanoma cell line, SK-MEL-5, showed high expression for TPC2 but not the TPC1 isoform on a transcript level and was selected for further investigation. Indeed, in wild-type (WT) SK-MEL-5 cells, robust Ca2+ responses were evoked that were twice as high as those observed in Hela cells. A TPC2 KO was created in this line to be used as a control for different experiments. As expected, TPC2-deficient SK-MEL-5 cells showed significantly reduced Ca2+ evoked responses. The simultaneous activation of TPC2 by TPC2-A1-P and TPC2-A1-N resulted in increased Ca2+ permeability and flux in WT cells; however, Na+ flux remained unaltered. Our study provides novel insight into the complex interaction of TPC2 with its ligands, demonstrating its preference for potentiating Ca2+ permeability over Na+ in response to signaling cues. This versatile behavior has profound implications on cellular function and physiology, particularly of significance when targeting TPC2 in disease models. In a pathological context, we expanded our gene expression analysis of endolysosomal cation channels and Rab proteins across various cancer types. We observed the highest expression for MCOLN1, TPCN2, and RAB7A particularly in comparison to other lysosomal genes tested, including MCOLN2, MCOLN3, TPCN1, and RAB7B. Intriguingly, these genes (MCOLN1, TPCN2, and RAB7A) showed the most significant enrichment in melanoma and a hepatocellular carcinoma line, surpassing other cancer types like cervical adenocarcinoma, ovarian cancer, colon adenocarcinoma, lung adenocarcinoma, and pancreatic ductal adenocarcinoma, among others. Consequently, melanoma, a highly aggressive type of skin cancer originating from melanocytes, was our primary focus of investigation. Identified risk factors for melanoma include excessive exposure to ultraviolet (UV) radiation, family history, fair hair, skin, and eye color. In particular, we focused on exploring the role of TPC2 in melanoma, given its substantial expression in this type of cancer, localization to mature melanosomes, as well as its pivotal role in pigmentation. Gain of function (GoF) mutations (G734E and M484L) in human TPC2 have been associated with reduced melanin production and blond hair. In accordance with this finding, our study focused on pigmented in vitro melanoma lines, MNT-1 (human) and B16F10 (mouse). Remarkably, the genetic ablation of TPC2 in MNT-1 demonstrated an increased melanin content and a larger but less acidic melanosome lumen. Besides, Naringenin, a natural flavonoid, has been found to block TPC2 activity. To expand on this discovery, we performed a screening of novel flavonoids derived from Dalbergia parviflora, which could potentially be more potent than Naringenin. Among the tested compounds, two were prominent hits: MT-8 (O-methylated isoflavone) and UM-9 (tri-O-methylated isoflavan). We validated these compounds using electrophysiology patch clamp experiments, which demonstrated their ability to inhibit TPC2 at a much lower concentration compared to Naringenin. In addition, both MT-8 and UM-9 showed the highest melanin generation in MNT-1 and B16F10 cells, indicating their potential as effective TPC2 antagonists. Accordingly, we sought to elucidate the physiological significance of TPC2 in regulating melanoma phenotypes in these pigmented lines using our hit compounds and the knockout model. Our experiments focused on assessing the impact of TPC2 on melanoma cell behavior, revealing substantial reductions in cell proliferation, migration, and invasion in the TPC2-deficient melanoma cells compared to their WT counterpart. We examined the downstream signaling cascades influenced by the endolysosomal machinery. Exceptionally, the melanoma oncogene, MITF, demonstrated a significant reduction on a protein level in the TPC2 KO MNT-1 cells compared to the WT cells. Further analysis using the protein stability cycloheximide chase experiments revealed that this downregulation was attributed to the proteasomal degradation of MITF. To confirm this observation, we treated the TPC2-deficient cells with the proteasomal inhibitor MG-132, which ameliorated MITF expression to levels comparable to WT cells. Moreover, we explored the signaling pathways known to regulate MITF and melanoma growth, including MAPK, cAMP, canonical Wnt, and Akt pathways. Our result demonstrated an inverse increase in GSK3β levels in TPC2 KO MNT-1 cells compared to WT, solidifying the role of GSK3β in melanoma as the negative regulator of MITF, promoting its proteasomal degradation. Intriguingly, a proteomic analysis of the TPC2 interactome unveiled Rab7 as an interaction partner of TPC2. Rab7a, a small guanosine triphosphatase (GTPase), serves as a lysosomal marker and plays critical roles in the trafficking and degradation of molecules, fusion of late endosomes and autophagosomes, and lysosomal biogenesis. Nevertheless, the functional impact of Rab7a on TPC2 channel activity and the consequent pathophysiological relevance of this interaction remains unclear. In our study (Abrahamian et al., 2023, appendix), we first reproduced the co-immunoprecipitation (Co-IP) data from Lin-Moshier et al., 2014 and further performed fluorescence resonance energy transfer (FRET) experiments, confirming the physical interaction between Rab7a and TPC2. Moreover, utilizing endolysosomal patch-clamp and Ca2+ imaging techniques, we demonstrated that Rab7a strongly enhances the activity of TPC2, establishing the functional interaction between these two lysosomal proteins. To explore the potential implications in melanoma, we generated different knockout models using CRISPR/Cas9, employed selective small molecule antagonists and agonists, and performed siRNA knockdown (KD) and overexpression (OE) studies in a range of melanoma lines with different mutational backgrounds. Interestingly, we observed significantly diminished melanoma cell proliferation, migration, and invasion in most MITF-dependent melanoma lines upon KO or KD of Rab7a or TPC2. However, most MITF-independent lines exhibited no alternations in melanoma phenotypes upon Rab7a or TPC2 depletion. Furthermore, we identified a positive correlation between the transcript levels of Rab7a and TPC2 in these melanoma lines, as well as a positive correlation between the protein expression of Rab7a with MITF and GSK3β. Consistent with the data obtained from the MITF-dependent pigmented MNT-1 line, the loss or pharmacological inhibition of Rab7a or TPC2 decreased the protein expression of MITF and β-Catenin, while GSK3β protein levels were increased. These findings corroborate the proposed model of the connection between the Wnt/β-Catenin pathway, MITF, and the endolysosomal machinery in melanoma20. In addition, we performed different rescue experiments in the Rab7a and TPC2 knockout lines. Our results demonstrated that the OE of Rab7a only partially alleviated the phenotype of TPC2 KO, whereas TPC2 OE effectively rescued the Rab7a KO phenotype. Based on these findings, we identified Rab7a as a melanoma oncogene and an effector of TPC2, highlighting their potential as targets for therapeutic interventions in melanoma. Overall, our findings advance our understanding of the therapeutic potential and interplay of lysosomal proteins in the context of neurodegenerative disorders and cancer, with a particular focus on breast cancer and melanoma. Through different works, we demonstrated that mutations, alterations in expression, or dysregulated activity of TRPML1 or TPC2 could result in detrimental effects on cellular functions and associated signaling cascades. Consequently, the investigation of endolysosomal cation channels and proteins and the generation of novel selective small molecule agonists and antagonists targeting these channels can offer valuable insights into the molecular mechanism underlying disease heterogeneity, shed light on the variations in disease manifestation, and uncovers novel opportunities for drug repurposing.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Lysosomes, melanoma, cancer, TPC2, TRPML1, Rab7, cancer, neurodegeration, endolysosomal system, channelopathies
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 18. März 2024
• 1. Bericht­erstatter:in: Grimm, Christian