A Mini Review
Background
One of the most common neurodegenerative cognitive disorders is Parkinson’s disease (PD) with seven to ten million patients worldwide. Environmental and genetic factors are known to be the root causes of this autonomic dysfunction. Several genetic mutations in SNCA (Synuclein alpha; PARK1), LRRK2 (Leucine-rich repeat kinase; PARK8), GBA (Glucocerebrosidase), PRKN (Parkin RBR E3 Ubiquitin Protein Ligase; PARK2), PINK1 (PTEN-induced kinase 1; PAR6), DJ1 (Protein deglycase DJ-1; PARK7), VPS35 (Vacuolar protein sorting-associated protein 35), EIF4G1 (Eukaryotic translation initiation factor 4 gamma 1), DNAJC13 (DnaJ (Hsp40) homolog, subfamily C, member 13), CHCHD2 (Coiled-coil-helix-coiled-coil-helix domain containing 2), α-SYN (alpha-synuclein), and UCHL1 (Ubiquitin C-terminal hydrolase L1) have been reported to be involved in the development of familial PD, among which the pathogenic G2019S mutation in LRRK2 (Leucine-rich repeat kinase II) is the critical contributor for neurotoxicity in 2% of all genetic cases (1-2). However, some recent studies have shown the activation of wild type LRRK2 in the case of idiopathic PD (iPD, 90% of all cases), irrespective of mutations. These observations make LRRK2 (with or without mutations) a potential therapeutic target for both types of PD (iPD and familial) (3). In addition to neurodegeneration, almost 20-32% of PD patients with LRRK2 mutations have been reported to develop cancer (2, 4-6). Though, both PD and cancer follow mechanistically different pathways, yet, in several studies on various cancers such as lung, colorectal, pancreatic, skin cancer, and leukemia, etc., LRRK2 is reported to be linked, indirectly or directly, either to cause toxic enhanced autophagy or to promote metastasis due to engagement in certain signaling pathways (7-9). This review article emphasizes the role of LRRK2 (mutant) as a risk factor to develop cancer in PD patients.
What is LRRK2 ?
LRRK2 enzyme is a type of ROCO-serine/threonine-specific kinase proteins, encoded by the LRRK2 gene. The enzymatic core of LRRK2 is comprised of Roc-COR (Ras-of-complex binding with C-terminal of Roc) and kinase (MAPKKK) domains mediating GTPase and kinase enzymatic functions, respectively (2, 10) (Figure-1). Also, besides, the four other domains are armadillo repeats (ARM), ankyrin repeats (ANK), leucine-rich repeats (LRR), and WD40 domain (hydrophilic domain) (2, 10). The genetic mutations in LRRK2 pockets such as in MAPKKK (G2019S, I2020T), LRR (I112V), Roc (N1437H, R1441C/G/H) and COR (Y1699C) are associated with PD. Among these, G2019S (Gly2019Ser) mutation leads to the increased phosphorylation of Ser1292, Ser910, and Ser935 in kinase, which in turns, phosphorylates a subgroup of Rab GTPases (Ras superfamily) in center of effector binding loop and results in neuroinflammation (late-onset PD) and enhanced autophagy (Figure-1) (2, 11-12). Autophagy is known to act as “good cop” in early phases of cancer initiation because it activates apoptosis of tumor cells, however, in later stages, it turns into “bad cop” by supplementing cancer cells with required nutrients to survive in stressful hypoxia-induced oxidative environment (13-14).
Among all Rab GTPases, Rab29, along with vesicular trafficking regulator i.e. VP35 (Vacuolar protein sorting-associated protein 35), acts as an upstream adaptor for LRRK2 because, after phosphorylation, it activates the LRRK2 kinase activity via interaction with LRRK2-ANK domain (11). In contrast, other phosphorylated Rabs such as Rab8/10/12/35 are downstream regulators of LRRK2 leading to ciliogenesis deficits (Rab 8, 10, 12, via interaction with RILP1/2), centrosomal defects (Rab 8a, centrosomal amplification), lipid storage (Rab 8a, enlargement of lipid droplets), α-synuclein aggregation (Rab 35) and delay in early / late endosomal trafficking (via Rab 5 to Rab 7 transition) (15-19).
In the past decade, the blockade of LRRK2-G2019S overexpression by using potential inhibitors (mostly kinase inhibition by competing with ATP for binding site either via DFG-in or DFG-out conformations) (20) has been investigated as a promising therapeutic approach for PD. To serve this purpose, different mechanisms were adopted like obstruction of GTP binding (Roc, essential for protein kinase activity), dimerization inhibition (COR), blockade of protein-protein interactions (ARM, ANK, and WD), stabilization of 14-3-3 interaction (LRR), and direct kinase inhibition (kinase) (21).
Figure-1: Structure of LRRK2; showing substrates (in Red) and inhibition of LRRK2 overexpression (in Green)
Signaling Pathways of LRRK2 in Oncology
However, in the past few years, research interest in evaluating the potential role of LRRK2 in various cancer types has been emerging. It is shown that exertion of autophagy, as well as metastasis via different signaling pathways (MEK/ERK/MAPK or JNK or JAK/STAT3 or AKT-4E-BP1), might be due to the increased kinase activity (structurally similar domain as in oncogene B-RAF) in G2019S-LRRK2 (5, 8, 22-23). In this regard, receptor tyrosine kinases (RTKs) play an essential role in oncogenic signaling via crosstalk with other cascades such as epidermal growth factor receptor (EGFR) (Figure-2). It has been identified previously that the downregulation of overexpressed LRRK2 in papillary renal cancer cells selectively blocked the mTOR signaling down to STAT3 in papillary renal carcinoma and inactivation of the JNK pathway in thyroid cancer cells, resulting in cell apoptosis and cell arrest (24-26).
Another kinase i.e. DCLK1 (Doublecortin-CaM-like kinase 1, MAP) has been reported as a potential marker of cancer stem cells (CSCs) in the lung, breast, colorectal and pancreatic carcinoma (7, 9, 27-29). Its overexpression is responsible to maintain cancer stemness (specific for CSCs) and to promote aggressiveness, cancer initiation, metastasis and angiogenesis. In addition to carcinomas, it is also involved in neurogenesis and neuronal migration. Though, the direct pathway-connection of LRRK2 with DCLK1 is still unknown, yet, it can be proposed (Figure-2) that upregulation of LRRK2-G2019S interacts either directly as with GSK3β or engages with signaling pathways such as JNK or ERK1/2 (MAPK) leading to epithelial-mesenchymal transition (EMT) due to elevated EMT-transcription factors such as Twist1/2, ZEB1/2, Slug and Snail (30-31). This transition results in degrading extracellular matrix (ECM) via proteolysis resulting in autophagy which promotes metastasis and survival of tumor cells (14, 28).
Inhibitors of LRRK2-G2091S have shown the upregulation of epithelial markers such as E-cadherin (CDH1) and the downregulation of transcription factors for EMT resulting in tumor cell apoptosis (7, 9). In pancreatic cancer cell lines, the overexpression of DCLK1 phosphorylated the focal adhesion kinase (FAK, a cytoplasmic tyrosine kinase) which mediated EMT via MAPK signaling pathway (9, 28), however, LRRK2-IN-1 inhibited DCLK1 overexpression resulting in the loss of EMT transcription factor i.e. Slug (9) (Figure-2). In a recent study, LRRK2-G2019S inhibitors have also shown an activation of the WNT signaling pathway (canonical) leading to the neuro-regeneration (32). In breast cancer patients, elevated levels of β-catenin have been observed which may be attributed to either mutation in β-catenin or decreased activity of the WNT pathway (non-canonical) (33). Such accumulation of β-catenin results in excessive tumor cell proliferation (33). Based on these studies, it can be proposed that LRRK2-G2019S upregulation may result in the overexpression of WNT which in turn leads to metastasis (Figure-2).
Figure-2: Reported and proposed signaling pathways of LRRK2 in cancer
In DNA damage response pathway (ATM-Mdm2-p53), LRRK2 plays the role of a downstream target because the overexpression, as well as the knockdown of LRRK2, showed no effect on ATM phosphorylation, however, it induced Mdm2 phosphorylation resulting in overexpression of p53 and p21, resulting in protection from abnormal cell proliferation (34) (Figure-2). Not only this, but a motif within the N-terminal of ARM-LRRK2 domain is connected to the extrinsic apoptosis pathway by mediating the binding with Fas-associated protein with death domain (FADD). The high phosphorylation LRRK2 due to G2019S leads to the downstream activation of FADD which in turn recruits mitochondrial co-factors (Bid/Bax) initiating a death cascade machinery (Caspase-3) in neurons (35) (Figure-2).
In cancer, phosphorylated FADD plays a critical role in apoptosis and cell arrest, and loss of FADD can be advantageous to cancer cell proliferation (36). On other hand, it is highly upregulated in ovarian and small cell lung carcinomas (37-38) and highly phosphorylated FADD may lead to the activation of nuclear factor kappa B (NF-κB) pathway which is antiapoptotic (39). However, the direct link between LRRK2-G2019S and FADD is missing in cancer study, but we can hypothesize that engagement of highly phosphorylated LRRK2-G2019S in JNK signaling probably can also lead to the overexpression of FADD which in turn, will lead to metastasis due to NF-κB activation (39) (Figure-2).
Future Aspects
In summary, the direct / indirect link of LRRK2 overexpression in mediating EMT leading to the toxic enhanced autophagy in cancer is a remarkably effective tool to be explored more in the future. It can also be proposed that LRRK2 inhibitors can provide a therapeutic insight, especially in the case of cancer metastasis, because of effective inhibition of DCLK1 (stem cell marker which is overexpressed in various cancer types and is responsible of promoting metastasis such as in pancreatic and breast cancer (9, 28) (Figure-3).
Figure-3: Proposed strategy to target cancer stem cells (CSCs) using LRRK2 inhibitors
The conventional therapy, such as chemotherapy, uses drugs which target only differentiating cells (the bulk of the tumor pool, formed by cancer stem cells (CSCs)). But cancer stem cells (CSCs, a fraction of tumor pool) are left untouched and cause relapse of cancer after months or years by regenerating tumor cells. Thus, the best therapeutic strategy may be the combination of both i.e. targeting both differentiating and cancer stem cells (CSCs). The role of LRRK2 inhibitors in this area is worth exploring for solid tumors, especially in case breast and ovarian cancer metastasis (because the efficacy of the most advanced and engineered therapeutic tool, i.e. CAR T cells (40-41), has failed for solid tumors due to incapability of infiltration in cancer cells).
References from Literature
1. Agalliu I, Ortega RA, Luciano MS, Mirelman A, et al. Cancer outcomes among Parkinson's disease patients with leucine-rich repeat kinase 2 mutations, idiopathic Parkinson's disease patients, and nonaffected controls. Mov Disord., 2019; 34(9): 1392-1398. [CrossRef]
2. Alessi DR, Sammler E. LRRK2 kinase in Parkinson's disease. Sci., 2018; 360(6384): 36-37. [CrossRef]
3. Di Maio R, Hoffman EK, Rocha EM, Keeney MT, et al.LRRK2 activation in idiopathic Parkinson's disease. Sci Transl Med., 2018; 10(451). pii: eaar5429. [CrossRef]
4. Saunders-Pullman R, Barrett MJ, Stanley KM, Luciano MS, et al. LRRK2 G2019S mutations are associated with an increased cancer risk in Parkinson disease. Mov Disord., 2010; 25(15): 2536-41. [CrossRef]
5. Agalliu I, San Luciano M, Mirelman A, Giladi N, et al. Higher frequency of certain cancers in LRRK2 G2019S mutation carriers with Parkinson disease: a pooled analysis. JAMA Neurol., 2015; 72(1): 58-65. [CrossRef]
6. Warø BJ, Aasly JO. Exploring cancer in LRRK2 mutation carriers and idiopathic Parkinson's disease. Brain Behav., 2017; 8(1): e00858. [CrossRef]
7. Weygant N, Qu D, Berry WL, May R, et al. Small molecule kinase inhibitor LRRK2-IN-1 demonstrates potent activity against colorectal and pancreatic cancer through inhibition of doublecortin-like kinase 1. Mol Cancer., 2014; 13: 103. [CrossRef]
8. Yakhine-Diop SMS, Rodríguez-Arribas M, Gómez-Sánchez R, Pizarro-Estrella E, et al. Chapter 5 - G2019S mutation of LRRK2 increases autophagy via MEK/ERK pathway. Autophagy, 2016; 9 (Human diseases and autophagosome): 123-142. [CrossRef]
9. Ikezono Y, Koga H, Akiba J, Abe M, et al. Pancreatic neuroendocrine tumors and EMT behavior are driven by the CSC marker DCLK1. [CrossRef]
10. Berwick DC, Heaton GR, Azeggagh S, Harvey K. LRRK2 Biology from structure to dysfunction: research progresses, but the themes remain the same. Mol Neurodegener., 2019; 14(1): 49. [CrossRef]
11. Seol W, Nam D, Son I. Rab GTPases as physiological substrates of LRRK2 kinase. Exp Neurobiol., 2019; 28(2): 134-145. [CrossRef]
12. Ysselstein D, Nguyen M, Young TJ, Severino A, et al. LRRK2 kinase activity regulates lysosomal glucocerebrosidase in neurons derived from Parkinson's disease patients. Nat Commun., 2019;10(1): 5570. [CrossRef]
13. Yun CW, Lee SH. The roles of autophagy in cancer. Int J Mol Sci., 2018; 19(11): 3466. [CrossRef]
14. Chen HT, Liu H, Mao MJ, Tan Y, et al. Crosstalk between autophagy and epithelial-mesenchymal transition and its application in cancer therapy. Mol Cancer., 2019; 18(1): 101. [CrossRef]
15. Roosen DA, Cookson MR. LRRK2 at the interface of autophagosomes, endosomes and lysosomes. Mol Neurodegener., 2016; 11(1): 73. [CrossRef]
16. Bae EJ, Kim DK, Kim C, Mante M, et al. LRRK2 kinase regulates α-synuclein propagation via RAB35 phosphorylation. Nat Commun., 2018; 9(1): 3465. [CrossRef]
17. Madero-Pérez J, Fdez E, Fernández B, Lara Ordóñez AJ, et al. Parkinson disease-associated mutations in LRRK2 cause centrosomal defects via Rab8a phosphorylation. Mol Neurodegener., 2018; 13(1): 3. [CrossRef]
18. Yu M, Arshad M, Wang W, Zhao D, et al. LRRK2 mediated Rab8a phosphorylation promotes lipid storage. Lipids Health Dis., 2018; 17(1): 34. [CrossRef]
19. Lara Ordónez AJ, Fernández B, Fdez E, Romo-Lozano M, et al. RAB8, RAB10 and RILPL1 contribute to both LRRK2 kinase-mediated centrosomal cohesion and ciliogenesis deficits. Hum Mol Genet., 2019; 28(21): 3552-3568. [CrossRef]
20. Estrada AA, Liu X, Baker-Glenn C, et al. Discovery of highly potent, selective, and brain-penetrable leucine-rich repeat kinase 2 (LRRK2) small molecule inhibitors. J Med Chem., 2012; 55: 9416–9433. [CrossRef]
21. A) Malik N, Gifford AN, Sandell J, Tuchman D, Ding YS. Synthesis and in vitro and in vivo evaluation of [3H]LRRK2-IN-1 as a novel radioligand for LRRK2. Mol Imaging Biol., 2017; 19(6): 837–845. [CrossRef] B) Malik N, Kornelsen R, McCormick S, Colpo N, Merkens H, Bendre S, Benard F, Sossi V, Schirrmacher R, Schaffer P. Development and biological evaluation of [18F]FMN3PA and [18F]FMN3PU for leucine-rich repeat kinase 2 (LRRK2) in vivo PET imaging. Eur J Med Chem., 2021; 211: 113005. [CrossRef]
22. Kim EK, Choi EJ. Pathological roles of MAPK signaling pathways in human diseases. Biochim Biophys Acta., 2010; 1802(4): 396-405. [CrossRef]
23. Renjini AP, Titus S, Narayan P, Murali M, et al. STAT3 and MCL-1 associate to cause a mesenchymal epithelial transition. J Cell Sci., 2014; 127(Pt 8): 1738-50. [CrossRef]
24. Looyenga BD, Furge KA, Dykema KJ, Koeman J, et al.Chromosomal amplification of leucine-rich repeat kinase-2 (LRRK2) is required for oncogenic MET signaling in papillary renal and thyroid carcinomas. Proc Natl Acad Sci USA., 2011; 108(4): 1439-44. [CrossRef]
25. Manzoni C, Mamais A, Dihanich S, Abeti R, et al. Inhibition of LRRK2 kinase activity stimulates macroautophagy. Biochim Biophys Acta., 2013; 1833(12): 2900-2910. [CrossRef]
26. Jiang ZC, Chen XJ, Zhou Q, Gong XH, et al. Downregulated LRRK2 gene expression inhibits proliferation and migration while promoting the apoptosis of thyroid cancer cells by inhibiting activation of the JNK signaling pathway. Int J Oncol., 2019; 55(1): 21-34. [CrossRef]
27. Tao H, Tanaka T, Okabe K. Doublecortin and CaM kinase-like-1 expression in pathological stage I non-small cell lung cancer. J Cancer Res Clin Oncol., 2017; 143(8): 1449-1459. [CrossRef]
28. Liu H, Wen T, Zhou Y, Fan X, et al. DCLK1 plays a metastatic-promoting role in human breast cancer cells. Biomed Res Int., 2019; 2019: 1061979. [CrossRef]
29. Park SY, Choi JH, Nam JS. Targeting cancer stem cells in triple-negative breast cancer. Cancers (Basel)., 2019; 11(7). pii: E965. [CrossRef]
30. Olea-Flores M, Zuñiga-Eulogio MD, Mendoza-Catalán MA, Rodríguez-Ruiz HA, et al. Extracellular-signal regulated kinase: A central molecule driving epithelial-mesenchymal transition in cancer. Int J Mol Sci., 2019; 20(12): 2885. [CrossRef]
31. Morandi A, Taddei ML, Chiarugi P, Giannoni E. Targeting the metabolic reprogramming that controls epithelial-to-mesenchymal transition in aggressive tumors. Front Oncol., 2017;7: 40. [CrossRef]
32. Zaldivar-Diez J, Li L, Garcia AM, Zhao WN, et al. Benzothiazole-based LRRK2 inhibitors as Wnt enhancers and promoters of oligodendrocytic fate. J Med Chem., 2019; doi: 10.1021/acs.jmedchem.9b01752. [CrossRef]
33. Zhan T, Rindtorff N, Boutros M. Wnt signaling in cancer. Oncogene., 2017; 36(11): 1461–1473. [CrossRef]
34. Chen Z, Cao Z, Zhang W, Gu M, et al. LRRK2 interacts with ATM and regulates Mdm2-p53 cell proliferation axis in response to genotoxic stress. Hum Mol Genet., 2017; 26(22): 4494-4505. [CrossRef]