Protein palmitoylation in cancer: molecular functions and therapeutic potential

Protein S‐palmitoylation (hereinafter referred to as protein palmitoylation) is a reversible lipid posttranslational modification catalyzed by the zinc finger DHHC‐type containing (ZDHHC) protein family. The reverse reaction, depalmitoylation, is catalyzed by palmitoyl‐protein thioesterases (PPTs), including acyl‐protein thioesterases (APT1/2), palmitoyl protein thioesterases (PPT1/2), or alpha/beta hydrolase domain‐containing protein 17A/B/C (ABHD17A/B/C). Proteins encoded by several oncogenes and tumor suppressors are modified by palmitoylation, which enhances the hydrophobicity of specific protein subdomains, and can confer changes in protein stability, membrane localization, protein–protein interaction, and signal transduction. The importance for protein palmitoylation in tumorigenesis has just started to be elucidated in the past decade; palmitoylation appears to affect key aspects of cancer, including cancer cell proliferation and survival, cell invasion and metastasis, and antitumor immunity. Here we review the current literature on protein palmitoylation in the various cancer types, and discuss the potential of targeting of palmitoylation enzymes or palmitoylated proteins for tumor treatment.


Introduction
Tumorigenesis is characterized by persistent cell proliferation, resistance to cell death, sustained angiogenesis, and increased cell invasion and metastasis. These features are accompanied by genome instability and mutation, cellular metabolism, replicative immortality, sustained inflammation, evasion of growth suppressors, and immune suppression [1]. The above processes are often controlled by various oncogenes and tumor suppressors, many of which are modified by posttranslational modifications (PTMs), such as phosphorylation, acetylation, ubiquitination, and palmitoylation [2,3].
Protein palmitoylation is a reversible lipid modification, through which palmitate, a 16-carbon palmitic acid, is added to a cysteine (Cys) residue via a thioester bond [4]. Palmitate is converted from fatty acids by fatty acid synthase (FASN) [5]. This process is initiated by taking up glucose in hepatocytes, followed by glycolysis to generate pyruvate; the latter is then catalyzed by pyruvate dehydrogenase to generate acetyl-CoA in mitochondria. Acetyl-CoA carboxylase then catalyzes acetyl-CoA to malonyl-CoA. Finally, FASN exerts its multifunctional enzyme activity to catalyze acetyl-CoA and malonyl-CoA to form palmitic acid in multiple catalytic steps (Fig. 1).
Numerous proteins, including key oncogenes and tumor suppressors, are palmitoylated, and their palmitoylation seems to be closely associated with tumorigenesis and tumor progression (Fig. 3) [13,15,17]. Despite some recent reviews summarizing the functions Fig. 1. Schematic diagram of fatty acid metabolism to generate palmitic acid. Glucose is taken up by hepatocytes to generate pyruvate through glycolysis, and pyruvate is then catalyzed by pyruvate dehydrogenase in mitochondria to generate acetyl-CoA. Next, acetyl-CoA carboxylase catalyzes acetyl-CoA to malonyl-CoA. Finally, FASN exerts its multifunctional enzymatic activity to catalyze the formation of palmitic acid from acetyl-CoA and malonyl-CoA in multiple catalytic steps. of ZDHHCs or PPTs in cancers [6,[18][19][20], a comprehensive review of this topic, particularly one focusing on the specific effects of protein palmitoylation across the various tumor types, is still lacking. Here we summarize recent findings on how protein palmitoylation affects key cancer hallmarks, including cell proliferation and survival, cell invasion and metastasis, and immune regulation in breast cancer, prostate cancer, gastrointestinal cancers, hematological cancers, melanoma, lung cancer, ovarian cancer, glioblastoma, and other tumor types. Moreover, we review tumor typespecific patterns versus protein palmitoylation functions that are shared across cancers, and discuss how palmitoylation can be specifically targeted for cancer treatment in a case-by-case scenario.

Cancer cell proliferation and survival
Palmitoylation could have a direct impact on substrate activity and regulate tumor growth. It has been presented that palmitoylation of phosphatidylinositol 4kinase IIa (PI4KIIa) simply accelerates tumor growth in mice by regulating its catalytic activity and subcellular localization [21,22] (Fig. 2, Table 1), and importantly, a small-molecule inhibitor of PI-273 that targets the palmitoylation insertion and activation loop of human PI4KIIa exhibits a significant inhibitory effect on breast cancer cell growth in vitro and in vivo [23] (Table 1). Similarly, palmitoylation also benefits tumor cells by inhibiting apoptosis: G-protein coupled receptor (GPCR) neurotensin receptor-1 (NTSR-1) was found to be dually-palmitoylated at Cys381 and Cys383 in MDA-MB-231 breast cancer cells, and mutation of the palmitoylation sites reduced the interaction between NTSR-1 and Gaq/11, and decreased the localization of NTSR-1 to the structured membrane microdomain, where Gaq/11 is preferentially present, impaired NTS-mediated ERK1/2 stimulation, and the ability to rescue cells from apoptosis induced by serum deprivation [24].

Cancer cell invasion or metastasis
Palmitoylation is also suggested to be involved in controlling metastasis in breast cancer, either promoting or inhibiting. For instance, palmitoylation of CD44 at Cys286 and Cys295 inhibited cell migration by promoting the lipid raft affiliation, and abrogating palmitoylation by mutating the palmitoylation site reduced CD44 raft localization, increased CD44-ezrin interaction, and improved invasive MDA-MB-231 cell migration [25]. On the contrary, the palmitoylation of integrin b4 (ITGb4) at cysteines Cys732, Cys736, Cys738, Cys739, and Cys742 by ZDHHC3 maintains its level in lipid rafts and promotes the invasive ability of breast cancer cells [26]. Intriguingly, curcumin, a natural polyphenol component of Curcuma longa, effectively inhibited breast cancer cell invasion by blocking autopalmitoylation of ZDHHC3, which regulates ITGb4 palmitoylation [26]. As angiogenesis brings nutrients and oxygen, it is conducive to tumor cell metastasis. KAI1/CD82 is a tumor metastasis suppressor in various cancers without affecting tumor formation [27]; it is significantly downregulated in estrogen receptor (ER)-positive breast cancer, and the ER antagonist fulvestrant was able to reverse ERmediated gene repression, induce significant KAI1/ CD82 upregulation, and inhibit breast cancer cell proliferation and migration [28]. Interestingly, ZDHHC4mediated palmitoylation localizes KAI1/CD82 to the cell membrane surface and induces the production of leukemia inhibitory factor through the Src/p53 pathway [16]. This in turn inhibits angiogenic factors in pericytes and endothelial cells themselves, thereby preventing angiogenesis and tumor progression [16] ( Fig. 2, Table 1). Limitations: many of these studies are performed only in vitro (cancer cell lines); more evidence from xenografts or in vivo tumor models will strengthen these claims.

Cancer inflammation or tumor immunity
In connection with immune-response, ZDHHC9mediated palmitoylation serves a pivotal role in regulating the stability of programmed cell death 1 ligand 1 (PD-L1), which binds to programmed cell death protein 1 (PD-1) on T-cells and transmits immunosuppressive signals [29]. Interestingly, the inhibition of PD-L1 palmitoylation by mutating the palmitoylation site or knocking down of palmitoylation enzyme with shRNA sensitized breast cancer cell lines to T-cell killing, thereby suppressing tumor growth in mice [30].

Cancer metabolism
Recent studies have shown that de novo synthesized fatty acids, including palmitate, benefit tumor cells [31]. Signaling through the plasma membrane epidermal growth factor receptor (EGFR, pmEGFR) and mitochondrial EGFR (mtEGFR) is often overactivated in cancer, and EGF-activated pmEGFR has been reported to increase FASN activity, and

Cancer cell proliferation and survival
Many reported palmitoylated proteins related to prostate cancer are involved in regulating cell proliferation or survival. Src family kinases Src, Fyn, or Lyn act as major convergence points for numerous receptors and cell-autonomous signaling pathways, resulting in enhanced cell proliferation, and metastatic potential during cancer progression [33,34]. Palmitoylated Src and Fyn have similar roles in regulation of clone formation of prostate cells. Palmitoylation of mutated Src (Serine 3 and Serine 6 were mutated to Cys) inhibited Src activation and led to suppressed clonogenicity, whereas a lack of palmitoylation in mutated Fyn (where Cys3 and Cys6 were mutated to Serine) showed dramatically increased clonogenicity [35]. Yet these studies did not elucidate the underlying molecular mechanisms downstream of Src or Fyn palmitoylation for their functions. Androgen receptor (AR) has a vital role in prostate cancer [36]. When the hormonal ligands testosterone and 5-dihydrotestosterone bind to AR, it dissociates from accessory proteins and transfers to the nucleus, triggering the expression of genes involved in cell proliferation and evasion of apoptosis, thereby boosting prostate cancer development [37]. A novel AR splice variant, AR8, was found to be localized to the plasma membrane via palmitoylation at Cys588 and Cys560, and overexpression of AR8 reduced prostate cancer cell proliferation and enhanced apoptosis in androgen-depleted culture conditions [38] (Fig. 2, Table 1). Although the molecular machinery in reaction to AR signaling still remains in mystery, another study did briefly suggest that upon androgen treatment, the levels of eukaryotic translation initiation factor 3 subunit L (eIF3L), Rab7a, and a-tubulin palmitoylation are elevated, which are required for the proliferation of prostate LNCaP cells [39,40].

Cancer cell invasion or metastasis
A study published in 2004 showed that the cancer metastasis suppressor KAI1/CD82 was palmitoylated at multiple sites (Cys5, Cys74, Cys83, Cys251, and Cys253) to inhibit migration and invasion of prostate cancer cells; and palmitoylation deficiency by mutating palmitoylation sites resulted in the loss of this inhibitory effect [41]. This study demonstrated a role for protein palmitoylation in the inhibition of prostate cancer progression.

Cancer metabolism
In immortalized prostate epithelial cells, overexpression of FASN activated b-catenin via Wnt1 palmitoylation, and subsequently activated ligand-independent AR [42]. Furthermore, FASN was reported to be transiently associated with lipid rafts, where it interacted with palmitoylated Caveolin-1, thereby contributing to prostate cancer progression [43]. Therapeutically, it was demonstrated that the knockdown of FASN with shRNA reduced intracellular palmitate levels, thereby inhibiting palmitoylation of the atypical GTPase Ras homolog family member U (RhoU). RhoU palmitoylation inhibition resulted in a significant loss of cell division cycle 42 (Cdc42) expression in 1542-CPTX primary prostate cancer cell line, ultimately increasing cell adhesion and inhibiting migration and invasion [44]. While FASN can boost the synthesis of fatty acids, including palmitate, and upregulate palmitoylation of potential different substrates, one important question here is: What are these substrates and how would these palmitoylated molecules coordinate in response to FASN signaling?

Cancer cell proliferation and survival
Palmitoylation was shown to be closely related to the progression of colorectal cancer (CRC) [45,46].
Stimulation of 17b-estradiol (E2) facilitates palmitoylationdependent membrane localization of ERb and its binding to Caveolin-1 and p38, thereby promoting apoptosis by p38/MAPK pathway activation in human colon adenocarcinoma DLD-1 cells [45]. However, contradictory results were also reported that the palmitoylation of ERb or the inhibition of p38/MAPK signaling promoted colorectal cancer cell growth [46]. Hence, the precise roles of ERb   palmitoylation and related downstream signaling cascades require further verification. Interestingly, more evidence seems to support that palmitoylation enhances apoptosis in CRC, the death receptors 4 (DR4) palmitoylation enhanced its lipid rafts localization in oxaliplatin resistance CRC cell lines, which enhanced tumor necrosis factorrelated apoptosis-inducing ligand (TRAIL) sensitivity and bind to DR4, thereby inducing cell apoptosis [47,48]. Likely, another death receptor, Fas (also termed CD95), was palmitoylated by ZDHHC7 at Cys199 for increasing its stability and lipid raft localization, and inhibition of Fas palmitoylation by knocking down of ZDHHC7 with siRNA promotes CRC cell lines to escape from FasLinduced cell death [49]. Remaining questions are how the dynamicity of palmitoylation is maintained in different scenarios and why that is important for regulating apoptosis by varied mechanisms? Wnt signaling has a key role in tumorigenesis, including CRC progression [50]. Of note, Wnt2B was shown to be palmitoylated, and Wnt2B palmitoylation alters its cellular localization, thereby indirectly influencing Wnt signaling. Furthermore, the level of Wnt2B palmitoylation in mitochondria is inversely correlated with intestinal tumorigenesis [51]. These findings implied that raising the palmitoylation levels of Wnt2B might bring beneficial effects in CRC.

Cancer cell invasion or metastasis
The palmitoylation of SFK family member YES plays an important role in regulating the activation of the RAS/MAPK signaling pathway. Mechanistically, the palmitoylation of YES at the SH4 domain regulates its localization in the cholesterol-rich membrane microdomain, which augmented the phosphorylation of EGFR, SHC, and SHP2, the upstream regulators of RAS/MAPK signaling, thereby inhibiting colon carcinoma cell adhesion and promoting invasion [52]. Akin to YES palmitoylation, palmitoylated claudin7 (cld7) was also reported to regulate cell motility. Specifically, palmitoylation facilitates cld7 to localize to the glycolipid-enriched membrane (GEM) domain, and mutating the palmitoylation site of cld7 reduces motility and invasiveness of rat pancreatic adenocarcinoma cells due to the possible mechanism that cld7 palmitoylation is involved in regulating the interactions with various components of vesicle transport machineries engaged in exosome biogenesis [12] (Fig. 2, Table 1). In agreement, another independent study indicated that tumor exosomes secreted by cancer-initiating cells, containing GEM-localized cld7, promote tumor cell dissemination and metastatic growth [53].

Cancer inflammation or tumor immunity
PD-L1 is specifically palmitoylated at Cys272 by ZDHHC3 in human colorectal cancer cell lines; inhibiting such palmitoylation promotes ubiquitinmediated protein degradation of PD-L1, thereby activating antitumor immunity and significantly suppressing tumor growth [17]. Accordingly, a competitive inhibitor (CPP-S1 peptide) of PD-L1 palmitoylation was developed, which reduces PD-L1 expression in tumor cells and enhances T-cell immunity against MC38 tumor [17]. Related to the theme of manipulating immunity by palmitoylation, IFNc receptor 1 (IFNGR1) was reported to be palmitoylated at Cys122, which then increased its interaction with AP-3 complex subunit delta-1 (AP3D1) upon the removal of optineurin, resulting in palmitoylated-IFNGR1 being sorted into lysosomes for destruction, ultimately suppressing T-cell-immunity and impairing immunotherapy efficacy [54] (Fig. 2, Table 1).

Protein palmitoylation and hematological cancers
Hematological cancers are commonly known as blood cancers, which usually form in the bone marrow or cells of the immune system. Although called blood cancer, hematological cancers include cancers such as a wide range of myelomas, lymphomas, and leukemias.

Cancer cell proliferation and survival
The RAS family members HRAS, NRAS, and KRAS were the first oncogenes identified in human cancers [55]. A 2018 study focusing on Wogonoside-a flavonoid extracted from Scutellaria baicalensis Georgi with antileukemic properties-reported that Wogonoside inactivates the NRAS/RAF1 signaling pathway by blocking NRAS palmitoylation in acute myeloid leukemia (AML) cells [56]. Of note, Wogonoside was also shown to promote depalmitoylation of yet another target, namely of phopospholipid scramblase 1 (PLSCR1) via targeting APT1 [56]. A second study confirmed that the association of NRAS with the plasma membrane requires palmitoylation at Cys181, and removal of palmitoylation was found to inactivate multiple signaling pathways downstream of oncogenic NRAS, ultimately suppressing leukemogenesis [57]. KRAS4A was also reported to be modified by palmitoylation at Cys180 [58]. Interestingly, leukemia could still be induced in mice expressing palmitoylation-deficient KRAS4A, albeit with slower kinetics. Unexpectedly, simultaneous mutation of the palmitoylation site and KIKK motif of KRAS4A abolished neurogenesis [59] (Fig. 2, Table 1). Last, ZDHHC6-mediated palmitoylation at Cys563 restrained cell surface localization of internal tandem duplication within FMS-like tyrosine kinase 3 (FLT3-ITD) protein, inhibited the activation of AKT and ERK, and leukemia cell growth [60].
Moreover, another study showed that the alkyl phospholipid 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC), which specifically induces apoptosis in leukemia cells by targeting the high cholesterol domain in cell membranes [61], lowered palmitoylation of the linker for activation of T-cells family member 2 (LAT2) at Cys25 and Cys28 in a lipid-raft-enriched fraction of leukemic cells, thereby promoting LAT2 degradation by proteasome, and hence decreasing cell proliferation and enhancing cell sensitivity to ODPC, perifosine, and arsenic trioxide [62]. Antigen-induced B-cell receptor (BCR) activation triggers palmitoylation of the E3 ubiquitin ligase Fbox protein 10 (FBXO10) at Cys49, Cys52, Cys180, Cys430, and Cys953, which causes FBXO10 to localize on the cell membrane to ubiquitinate and degrade the human germinal center-associated lymphoma (HGAL) protein.
HGAL downregulation then prevents the uncontrolled BCR signaling that is linked to development of lymphoid hyperplasia, and lymphomagenesis [63] (Fig. 2, Table 1). The shortcomings of these studies are that they did not specify the enzymes catalyzing palmitoylation, and some conclusions lack support from palmitoylation-site mutant protein.

Cancer cell invasion or metastasis
As shown above, KAI1/CD82 palmitoylation prevents tumor metastasis in both breast and prostate cancers; however, the palmitoylation of KAI1/CD82 on the membrane of AML cells promotes the development of aggressive leukemia through recruiting and stabilizing protein kinase C alpha (PKCa) in membrane clusters, and subsequently sustaining ERK signaling [64]. Conversely, inhibiting KAI1/CD82 palmitoylation by mutating the palmitoylation site dramatically impairs the formation and organization of N-cadherin clusters, and subsequently diminishes bone marrow homing of AML [65] (Fig. 2, Table 1). The functional discrepancies of KAI1/CD82 palmitoylation in different types of tumors might cause problems if targeting KAI1/CD82 palmitoylation, or at least, one should be cautious under such circumstances.

Cancer inflammation or tumor immunity
It has been suggested that immune suppression is closely related to leukemogenesis, yet possibly mediated by diversified molecular machineries. Extended evidence suggests that the uptake by monocytes of AML-derived extracellular vesicles with abundant palmitoylated proteins on their surface promotes the differentiation of myeloid-derived suppressor cells, thereby enhancing immune escape [66] (Fig. 2, Table 1).

Cancer cell proliferation and survival
The melanocortin-1 receptor (MC1R) is a member of the GPCR family that is expressed on melanocytes and enhances ultraviolet (UV) tolerance when activated [67], which reduces the risk of melanoma. However, mutational inactivation of MC1R (Mc1r RHC variants) results in reddened hair color, poorer skin tanning ability in humans, and increases the risk of melanoma [68]. Interestingly, ZDHHC13 was found to mediate MC1R palmitoylation at Cys78 and Cys315, and MC1R palmitoylation is required for activation of MC1R signaling, which leads to enhanced pigmentation, UV-induced cell cycle arrest, and control of melanomagenesis [15]. Furthermore, employing Palm-B to pharmacologically increase palmitoylation recovers the deficiencies of Mc1r RHC variants and inhibits melanomagenesis [15]. Two years later, another study demonstrated that APT2 is the depalmitoylation enzyme of MC1R, and ML349, a selective APT2 inhibitor, could significantly increase MC1R palmitoylation and the downstream signaling that prevents UVinduced melanomagenesis [69] (Fig. 2, Table 1).

Cancer cell invasion or metastasis
It was suggested that ZDHHC20 mutants in melanoma cells reduce palmitoylation of melanoma cell adhesion molecule (MCAM) and impair the MCAM ability to inhibit cell invasion, whereas Wnt5a promotes depalmitoylation of MCAM at Cys590 via phosphorylated APT1 [70]; APT1 inhibition with Palm-B prevents Wnt5a-induced depalmitoylation, asymmetric MCAM localization, and cell invasion [71]. Similar to leukemia, NRAS palmitoylation is required for pro-tumorigenic NRAS signaling in melanoma, which often carries NRAS activating mutations [72,73] (Fig. 2, Table 1). Unfortunately, in the past NRAS has often been deemed as "undruggable" due to the lack of hydrophobic binding pockets on the NRAS surface [74]. Fortunately, Vora et al. [75] developed an amphiphilemediated depalmitoylation (AMD) strategy using C8 alkyl cysteine, to cleave S-palmitoyl groups from endogenous membrane-associated NRAS protein, resulting in a significant reduction in NRAS palmitoylation, inhibition of NRAS signaling, as well as melanoma progression (Table 1).

Cancer cell proliferation and survival
In a study by Sanders et al. [76], who analyzed data from 15 palmitoylation proteomics studies, pancreatic ductal adenocarcinoma emerged as one of the top 5 diseases with enrichment of proteins that are known palmitoylation targets. This implies that aberrant palmitoylation may be involved in pancreatic tumorigenesis [76]. Published studies suggested that overexpression of cytoskeleton associated protein 4 (CKAP4) or LDL receptor-related protein 6 (LRP6) promotes pancreatic cancer progression [77,78]. Interestingly, CKAP4 was found palmitoylated by ZDHHC2 at Cys100, and LRP6 was palmitoylated at Cys1394 and Cys1399 (with palmitoylation enzymes not yet defined). Mechanistically, palmitoylation induces localization of CKAP4 and LRP6 in the detergent-resistant membrane (DRM) fractions, thereby activating the PI3K-AKT pathway and promoting cell proliferation [79]. Furthermore, a 2019 study demonstrated that depalmitoylation promotes the colocalization and direct interaction of KRAS4A and hexokinase 1 (HK1) on the outer mitochondrial membrane (OMM), which in turn promotes glucose consumption. Accordingly, disruption of KRAS4A reduces glucose consumption in pancreatic cancer cells [80]. Recently, it was also revealed that upregulation of long noncoding RNA plasmacytoma variant translocation 1 (PVT1) promotes the movement of multivesicular bodies towards the plasma membrane by regulating the colocalization of palmitoylated YKT6 at Cys194 and Cys195 and vesicle-associated membrane protein 3 (VAMP3), which then stimulates exosome secretion, and may serve a vital role in tumorigenesis [81] (Fig. 2, Table 1).

Cancer cell proliferation and survival
Non-small cell lung cancer (NSCLC) accounts for nearly 85% of all lung cancer cases that are clinically diagnosed [82]. The link between protein palmitoylation and NSCLC was established by the finding that ZDHHC21 palmitoylates EGFR at Cys1025, Cys1034, and Cys1122 in HEK293T cells, where EGFR and ZDHHC21 were coexpressed and purified for mass spectrometry; and inhibition of ZDHHC21 with shRNA in MDA-MB-231 cells or expressing the palmitoylation site mutation of EGFR plasmids in NIH 3T3 cells enhanced EGFR activation and increased EGFR signaling-dependent cell survival and migration [83]. Moreover, another independent study also showed that blocking EGFR palmitoylation resulted in inhibition of PI3K signaling by reducing the EGFR association to p85, ultimately suppressing mutant KRAS lung tumorigenesis [84]. While these studies pointed out the potential pharmaceutical value of targeting EGFR palmitoylation for treating NSCLC, interestingly, another group presented that blocking FASN with Orlistat (a reversible inhibitor of gastric and pancreatic lipases) treatment prevents EGFR palmitoylation, enhances ubiquitination and degradation of EGFR, and then inhibits NSCLC cell proliferation, and suppresses in vivo tumorigenesis [85] (Fig. 2, Table 1). Taken together, similar to other types of tumors, EGFR palmitoylation also plays a critical role in NSCLC progression.

Cancer metabolism
In advanced GBM, the membrane expression and palmitoylated form of solute carrier family 1 member 3 (SLC1A3, a glutamate transporter) were dramatically downregulated, resulting in impaired glutamate uptake [87] (Fig. 2, Table 1). However, the specific palmitoylation site in SLC1A3 and the enzymes regulating SLC1A3 palmitoylation have not yet been explored. To maintain rapid proliferation, even in the presence of oxygen, cancer cells absorb large amounts of glucose for glycolysis, a process known as the Warburg effect [88]. The glucose transport protein GLUT1 is a critical transmembrane protein to regulate glucose transport [89]. ZDHHC9-mediated GLUT1 palmitoylation at Cys207 maintains GLUT1 localization in the cell membrane for glucose transport. Knocking out ZDHHC9 using the CRISPR/Cas9 system or mutating the GLUT1 palmitoylation site abolished palmitoylation and cell membrane distribution of GLUT1, resulting in impaired glycolysis, cell proliferation, and GBM tumorigenesis [13] (Fig. 2, Table 1).

Cancer cell proliferation and survival
ZDHHC12 mediates palmitoylation of claudin 3 (CLDN3) at Cys103, Cys106, Cys181, Cys182, and Cys184 to enhance ovarian cancer progression and, reversely, defective CLDN3 palmitoylation inhibits membrane localization and protein stability of CLDN3, with a negative impact on ovarian cancer cell growth [90]. In addition, the palmitoylation of epithelial cell adhesion molecule (EpCAM) affects the formation of a complex among EpCAM, claudin isoforms, and KAI1/CD82, which is important in ovarian cancer progression, whereas 2-BP treatment inhibits EpCAMP-CLDN-KAI1/CD82 complex formation [91]. Moreover, ZDHHC18-medited palmitoylation of malate dehydrogenase 2 (MDH2) at Cys138 is essential for mitochondrial respiration and ovarian cancer cell proliferation both in vitro and in vivo, and loss of MDH2 palmitoylation by mutating the palmitoylation site or knocking down of ZDHHC18 with shRNA inhibited the clonogenic capability of ovarian cancer cells [14]. On the contrary, ZDHHC7-mediated palmitoylation of the tumor suppressor function of scribble (SCRIB) at Cys4 and Cys10 is important for regulating OVCAR8 cell proliferation and inhibiting the activation of YAP, MAPK, and PI3K/AKT pathways in MCF10A cells [92] (Fig. 2, Table 1). These examples strengthen the notion that palmitoylation regulates tumorigenesis in both directions.

Protein palmitoylation and other cancers
Even though hepatocellular carcinoma (HCC) is the most frequent primary liver cancer and a serious medical problem [95], there are limited studies relating protein palmitoylation to HCC progression. A report stated that high cholesterol levels enhance the lipid raft localization of CD44 in a palmitoylation-dependent manner, and disrupts CD44-Ezrin binding, ultimately reducing HCC cell migration and metastasis [96] (Fig. 2, Table 1). This finding is consistent with the reported role for CD44 palmitoylation in breast cancer [25].
Moreover, protein palmitoylation has been implicated in the development of renal clear cell carcinoma (RCC), bladder cancer, gastric adenocarcinoma, and osteosarcoma. The expression patterns of ZDHHC have been correlated with RCC prognosis, as well as with the immune profiles, molecular features, and signaling pathways of RCC [97]. In bladder cancer, increased palmitoylation levels of FASN and PD-L1 were correlated with cisplatin resistance [98]. However, it remains to be investigated whether a reduction in the palmitoylation levels of FASN and PD-L1 can sensitize bladder cancer cells to cisplatin. Besides, in the healthy bladder, ZDHHC2 has been reported to palmitoylate CKAP4 at Cys100, thereby inhibiting epithelial cell proliferation [99] (Fig. 2, Table 1).
Reduced ZDHHC2 expression has been linked to lymph node metastasis and is an independent predictor of poor prognosis in gastric adenocarcinoma [100] ( Table 1). In pituitary tumors, E2 treatment promoted ZDHHC7-and ZDHHC22-mediated palmitoylation of ERa at Cys477, enhanced plasma membrane ERa pools, and thus activated ERK1/2, thereby promoting tumor cell proliferation [101][102][103] (Fig. 2, Table 1). Taken together, all the above studies indicate that protein palmitoylation is deeply involved in regulating tumorigenesis in a wide range of varied cancer types.

Targeting protein palmitoylation in cancer
Since its discovery more than 40 years ago, scientists have gained a more detailed understanding of the biological role and regulation of protein palmitoylation. Abnormal palmitoylation status has been reported in several disease states, such as in neurodegenerative diseases [104,105], inflammation [106,107], bacterial and viral infection [108,109], and human cancers, as described in this review that the involvement of palmitoylation in the progression of almost all cancer types is reflected through the aberrant expression patterns of ZDHHCs and PPTs and through the changes in palmitoylation levels of cancer-associated proteins, which in turn affect their functions in tumor cell proliferation [39], adhesion [110], migration [111], metastasis [112], and apoptosis [113]. These findings raise the possibility that targeting of either ZDHHCs and PPTs or of palmitoylated cancer-associated proteins might benefit the treatment of varied cancer types, as discussed above.

Pan-cancer strategies
For targeting ZDHHCs or PPTs, shRNA or enzyme activity inhibitors were commonly used to either downregulate the protein expression or inhibit the enzymes functioning. For example, inhibiting PPT1 with DC661 also dramatically suppresses the development of a variety of tumors [114]. For targeting a specific palmitoylated protein, palmitoylation-competitive peptide or species (either small molecule or peptide) to block the specific palmitoylation site was introduced.

Breast cancer
ZDHHC5 regulated Flotillin2 palmitoylation is important for Flotillin2 localization in lipid rafts [115], which serve critical roles in carcinogenesis [116]. Furthermore, it showed that the liver X receptor (LXR) agonist T0901317 disrupted lipid rafts in breast cancer cells by downregulating Flotillin 2 and its membrane-associated palmitoylation enzyme, ZDHHC5, and the disruption of lipid rafts is linked to the antiproliferative effect of T0901317 [117]. Other examples raised the possibility to target the palmitoylation/depalmitoylation enzymes directly. ZDHHC3 is significantly elevated in both malignant and metastatic human breast cancer, and knockdown of ZDHHC3 with shRNA in MDA-MB-231 cells leads to oxidative stress and senescence, enhancing the recruitment of antitumor macrophages and natural killer cells associated with the clearance of senescent tumor cells, thereby reducing xenograft growth in both primary tumors and metastatic lung colonies [118]. Moreover, ZDHHC3 ablation with shRNA in combination with the anticancer drug, poly ADP-ribose polymerase (PARP) inhibitor PJ-34, increased oxidative stress and inhibited MDA-MB-231 cell proliferation [119]. Similarly, knockdown of depalmitoylating enzyme APT1 (also known as LYPLA1) with shRNA in MDA-MB-231 cells depleted a specific subpopulation of tumorigenic cells, resulting in colony formation being suppressed [120], which may   (Table 1). Although both ZDHHC3 and APT1 depletion seem to have a similar effect on preventing the proliferation of the same breast cancer cell (MDA-MB-231), the molecular mechanisms might be different, as ZDHHC3 regulates oxidative stress and senescence, while APT1 is involved in controlling apoptosis. Further understanding the underlying cause might rely on the identification of their substrates regarding that the opposite roles of ZDHHC3 and APT1 played in protein palmitoylation. Snail is a prominent inducer of epithelial to mesenchymal transition (EMT) and cancer progression [122].
Overexpression of Snail in MCF10A cells induces the expression level of APT enzymes and accelerates depalmitoylation, thereby affecting the palmitoylation cycle of numerous proteins, which may affect breast cancer cell polarity, EMT, and tumor suppression [123]. Taken together, these findings proved in principal that ZDHHC3 and APT1 are potential pharmaceutical targets for treating breast cancer.

Prostate cancer
ZDHHC3-mediated palmitoylation plays a key role in regulating the expression, stability, and function of integrin a6b4, and knockdown of ZDHHC3 with shRNA affects integrin-dependent cable formation and signaling in prostate tumor cells [124]. Moreover, ZDHHC14, as a tumor suppressor, is frequently downregulated in testicular germ cell tumors and prostate cancer, and overexpression of ZDHHC14 resulted in inhibited cell viability and promoted cell apoptosis [125] (Table 1). Last, but not least, blocking EGFR palmitoylation (mutation of Cys797, Cys1025, and Cys1122 to alanine) disrupted TKI-induced EGFR dimerization, resulting in the apoptosis of TKIresistant cancer cells [93].

Colorectal cancer
Cancer stem cells (CSCs) are types of tumor cells with self-renewal and multi-differentiation capabilities, which play a key role in tumor progression. One study illustrated that a small-molecule inhibitor, RU-SKI 43, targets SHH-palmitoylating Hedgehog acyltransferase (HHAT), and effectively reduces colon CSCs survival at low dosages by blocking all signaling downstream of SHH [126]. Surprisingly, another group showed that dual MEK-PI3K inhibitor therapy somehow significantly reduced KRAS mutated mucin 2 (MUC2) expression, MUC2 palmitoylation, and secretion in colon cells, ultimately suppressing mucinous tumor growth in vivo [127]. Last, but briefly, the overexpression of ZDHHC9 could dramatically suppress the proliferation of colorectal cancer cells [128] (Table 1). However, it should be noted that more evidence from xenograft mouse models and clinical verifications are still needed to validate these potentially important findings.

Hematological cancers
It was revealed that the universal palmitoylation inhibitor 2-Bromopalmitate (2-BP) and all-trans retinoic acid have a synergistic differentiation-inducing effect in acute promyelocytic leukemia. Mechanically, it was suggested that 2-BP covalently binds to retinoic acid receptor alpha (RARa) and prevents RARa degradation, resulting in the promoted transcription of RARatarget genes [129]. This suggests that 2-BP has potential in treating leukemia, although it should be noted that 2-BP lacks substrate specificity, and therefore might bring unexpected toxicity in vivo. In addition, the combination of the nonspecific depalmitoylation inhibitor palmostatin B (Palm-B) with gilteritinib (FLT3 inhibitor) also significantly suppressed FLT3-ITD-mediated signaling and leukemia progression [60], indicating that raising the level of palmitoylation might also bring beneficial effects in leukemia. On the side of PPTs, CD95, a member of the tumor necrosis factor superfamily, has been demonstrated to bind to its ligand (CD95L) and transmit a death signal to cells, causing apoptosis [130]. However, tumor cells escape CD95-mediated apoptosis by silencing CD95 expression [131]. Therefore, knockdown of APT1 and APT2 with siRNA or pharmacological inhibition of APT1 and APT2 using Palm-B, or overexpressing miRs-138/-424 to deregulate and target APT1 and APT2, restores CD95-mediated apoptosis in chronic lymphocytic leukemia cells [121]. Additionally, ABD957, a strong and selective covalent inhibitor of the ABHD17A/B/C, inhibits NRAS depalmitoylation in AML cells, resulting in suppressed NRAS signaling and the development of NRAS-mutant AML [132] (Table 1). These examples demonstrated that the regulatory roles of palmitoylation in hematological cancers can be bidirectional, and hence the targeting strategies should be designed carefully case-by-case.

Melanoma
It was reported that inhibition of PPT1 with specific PPT1 inhibitors hydroxychloroquine (HCQ) or dimeric CQ DC661 induced interferon-b secretion in macrophages and enhanced the antitumor efficacy of the anti-PD-1 antibody in melanoma [133]. In addition, a mono-palmitoylated peptide that contained the dominant MHC class-I and II epitopes of the human melanoma antigen gp100 (gp100 280-288/45-59 ) or the mouse model antigen ovalbumin (OVA 257-264/323-339 ) conjugated to palmitolate anhydride (C16:0) was developed that is efficiently internalized by DCs and subsequently cross-presented to CD8 + T-cells, ultimately inhibiting melanoma progression [134] (Table 1).

Pancreatic cancer
A potent antitumor compound, DQ661, has been identified, which specifically targets and inhibits PPT1 in lysosomes, resulting in rapid accumulation of palmitoylated proteins, thereby impairing mTOR and lysosomal catabolism, which in turn significantly inhibiting tumor growth in mouse models of melanoma, colorectal, and pancreatic cancer [135] (Table 1).

Lung cancer
A study illustrated that knockdown of ZDHHC5 dramatically inhibited cell proliferation, colony formation and cell invasion in vitro, as well as severely hindered NSCLC tumor xenograft formation, demonstrating the oncogenic capacity of ZDHHC5 [136]. Similarly, another study found that silencing APT1 with shRNA significantly reduced NSCLC cell proliferation, migration, and invasion in vitro [137] (Table 1). Disclosing the enzymatic substrates of ZDHHC5 and APT1 will be the key to understand why they play similar roles in lung cancer.

Glioblastoma
Similar to protein palmitoylation inhibitors, substrateanalog inhibitors (similar in structure to one of the substrates of the reaction they are inhibiting, e.g. 2-BP, cerulenin or tunicamycin) significantly suppressed GBM cell survival by inhibiting the cell cycle and promoting apoptosis [138] (Table 1). Interestingly, a study showed that local anesthetics could attenuate glioblastoma stem cell proliferation and self-renewal via diminishing ZDHHC15-mediated palmitoylation of interleukin 6 signal transducer (IL6ST, also named GP130) [139]. Yet, the molecular connections between local anesthetics and ZDHHC15 activity remain to be further examined.

Ovarian cancer
Two selective and potent FASN inhibitors, TVB-3166 and TVB-3664, were discovered and used in ovarian, lung, prostate and pancreatic cancer mouse models, which presented that inhibiting FASN in combination with taxane therapy inhibited tumor cell growth both in vitro and in vivo, possibly by disrupting tubulin palmitoylation, expression and microtubule organization [140]. Mechanistically, it is negotiable how exactly TVB-3166 exerts its antitumor effect as another study showed that TVB-3166 treatment alters ERa subcellular localization and reduces ERa levels by inducing endoplasmic reticulum stress, and exhibits antitumor activity in tamoxifen-resistant breast tumor cells [141] ( Table 1). Further investigations are warranted on this issue.

Other cancer types
For the treatment of HCC, GNS561, a new lysosomotropic small molecule drug was developed to target PPT1 to modulate lysosomal deacidification and inhibit mammalian target of rapamycin (mTOR) signaling pathway. Moreover, GNS561 and sorafenib (a multikinase inhibitor) were found to have a synergistic impact on preventing tumor development and cell proliferation in a mouse tumor model [142,143] (Table 1). In addition, the combination of adriamycin (an antibiotic drug) and 2-BP strongly inhibited the proliferation of osteosarcoma cell lines and primary osteosarcoma cells [144] (Table 1).

Future perspectives
First, the specific substrates for ZDHHCs and PPTs have not yet been fully identified. So far, the substrates of APT1 and PPT1 have been identified in the human embryonic kidney cell line HEK293T [145] and in the human neuroblastoma cell line SH-SY5Y [146], while some substrates of other ZDHHCs and PPTs have been reported [13][14][15]69,90,115,132,139,147,148]. Several studies using palm-proteomics (isolation of total palmitoylated proteins + mass spectrometry) have successfully identified a spectrum of substrates for ZDHHC3 in breast and prostate cancer cells [119], indicating that ZDHHCs and PPTs have multiple substrates, which also vary depending on cancer type. Moreover, in our experience, palm-proteomics analyses may give false-positive results, and further validation by Acyl-Biotin Exchange (ABE)/Acyl-RAC and pointmutagenesis are necessary. Interestingly, ZDHHC family members appear to differ from the protein structure perspective, and these differences may confer specificity in substrates recognition. For example, ZDHHC5 and ZDHHC8 possess a long and highly disordered C-terminal tail that can interact with structural aspects of many proteins, such as the PDZ domain; whereas ZDHHC17 and ZDHHC13 contain an ankyrin repeat domain, which enables them to interact with many known ZDHHC17 substrates, including huntingtin, SNAP25, and cysteine chain proteins [149]. Understanding this from such an angle might facilitate clarifying the substrates complexity of ZDHHCs/PPTs, although much effort is needed. Second, only a few selective inhibitors targeting ZDHHCs, PPTs, or palmitoylated proteins have been developed to date. As palmitoylation may regulate tumorigenesis in both directions (promoting or inhibiting cancer progression), palmitoylation-targeting strategies should be carefully designed according to each specific situation.
Initially, several studies reported that 2-BP was used to inhibit tumorigenesis at a cellular level [150]; however, the inhibition of protein lipidation is toxic to normal cells, which might limit the application of 2-BP in cancer patients. Furthermore, as both oncogenes and tumor suppressors can be palmitoylated, the application of 2-BP might compromise its treatment effect due to the lack of selectivity. Later on, Palm-B was developed to selectively target APT1, but eventually it was found to target APT2 as well. To specifically inhibit each of the two APTs, the selective APT1 and APT2 inhibitors ML348 and ML349 were developed. Recently, the ABHD17A/B/C inhibitor ABD957 as well as the PPT1 inhibitors DC661, DQ661, and GNS561 were also identified [69,114,132,133,135,142,143,151]. Yet the target specificities of these "newly" identified inhibitors need to be carefully validated.
No specific and potent inhibitors have so far been developed to target ZDHHCs. However, considering that ZDHHCs might have multiple and even crosssubstrates for palmitoylation [149], potential ZDHHCs inhibitors might face an unexpected outcome, and thus possibly reduce their translational values in clinics [6]. Alternatively, other strategies, such as developing smallmolecule inhibitors targeting palmitoylation insertions and AMD have been reported [23,75]. Indeed, a competitive inhibitor against specific sites of palmitoylation showed a good inhibitory efficacy in colorectal cancer [17]; additionally, an interesting vaccination strategy using mono-palmitic acid modified antigenic peptide that could significantly facilitate antitumor immunity in melanoma [134]. However, more successful examples are awaited to prove in principal that these strategies are truly effective both in vitro and in vivo.
Third, it is difficult to establish a direct mechanistic connection between palmitoylation of specific proteins and their function in cancer. This stems from the lack of specific palmitoyl transferase inhibitors, of palmitoyl-mimetic mutations, or of consensus palmitoylation sequences that make gain of function studies challenging. Moreover, there are no antibodies to palmitoylated epitopes, and therefore correlation between palmitoylation levels of a protein and tumor grade in patient samples are extremely difficult. It should be noted that most of the examples summarized here are in vitro studies in cell lines. Therefore, introducing a palmitoylation antibody and novel tools as specific agonist or antagonist of palmitoylation will greatly advance the research fields.
Together, it can be visualized that the functional studies of palmitoylation in tumorigenesis are mostly focused on breast cancer, prostate cancer, colorectal cancer, leukemia, melanoma, pancreatic cancer, and NSCLC, but are less explored in other types of cancer, including RCC, osteosarcoma, GBM, ovarian cancer, HCC, bladder carcinoma, gastric adenocarcinoma, and pituitary tumors. More pathogenic mechanisms related to protein palmitoylation are expected to be discovered in all these human cancer types, which will give a solid theoretical basis for therapeutic development in the future.