Cell proliferation is a cycle that consists of G1, S, G2 and M phases, which can usually be regulated by cyclin (cyclin A, B, D and E) and cyclin-dependent kinase (CDK; CDK1, 2, 4 and 6) protein families. Abnormal expression of these regulators can lead to uncontrolled cell proliferation, and then tumor development [45,46,47].

Cav-1 is involved in the lung cancer cell cycle. Typically, in a majority of NSCLC cell lines (H460 and A549), Cav-1 has oncogenic characteristics that facilitate lung cancer cell proliferation. In H460 cells, Cav-1 knockdown can reduce the expression of cyclin D1 and proliferating cell nuclear antigen (PCNA, a regulator of DNA replication during cell division). Subsequently, most cells pause at the G1/S stage, thus inhibiting cell proliferation [48]. In paclitaxel-resistant A549 cells, Cav-1 knockdown results in decreased proliferation by reducing cyclin D1 expression and inhibiting AKT phosphorylation. As a result, the dividing cells are arrested at the G0/G1 phase and the number of cells in the S or G2/M phase is dramatically reduced [49]. While in SCLC cell lines (SCLC-R1, H446), Cav-1 can play dual roles on regulating cell proliferation. The proliferation inhibition is found in Cav-1-knockdown SCLC-R1 and RAL cells. Due to Cav-1 downregulation, the cyclin D1 and CDK4 levels are both reduced. Meanwhile, the expression and phosphorylation of the signal transducer and activator of transcription 3 (STAT3), a key transcription factor targeting gene CCND1 (can encode cyclin D1), are also decreased. Reduced expression of cyclin D1 can eventually lead to slow cell division. These factors contribute to cell growth arrest all together [50]. Such cases indicate that Cav-1 knockdown can inhibit lung cancer cell proliferation via negatively regulating the cell cycle, which suggests a probably positive correlation between Cav-1 and lung cancer cell proliferation. However, Sun et al. drew a completely opposite conclusion in H446 cells. They found that Cav-1 over-expression could decrease pERK1/2 expression and make most cells arrest at the G2/M phase, and finally inhibit cell proliferation [27]. In the study, they also found that Cav-1 over-expression could lead to estrogen receptor (ER) and progesterone receptor (PR) reductions. Estrogen and progesterone have been reported to stimulate cell proliferation in breast cancer by elevating cyclin G1 expression [51]. However, the direct evidence of Cav-1-mediated cell proliferation by acting on ER and PR still lacks. This is probably the reason why this cell line behaves contrary to the others. Cav-1 can also facilitate lung cancer cell proliferation via other pathways. In A549 and GLC-82 cells, Cav-1 can function as one of the plasma membrane components to mediate EGFR endocytosis with the help of prostaglandin E2 (PGE2), to induce its nuclear translocation. Then EGFR can interact with STAT3 in the nucleus and promote STAT3 activation, leading to enhanced cell proliferation. Thus, Cav-1 ablation will impair EGFR accumulation within the nucleus and restrain cell proliferation [52]. Cav-1 over-expression can increase GLC-82 cell proliferation, and an obvious tumor growth was also observed in mice models transplanted with such cells [26]. In A549 and SK-MES-1 cells, by RT-qPCR, lncRNA HOTAIR expression was checked and shown to be increased by Cav-1. Then, as was shown in the results of CCK-8 and transwell assays, they can synergistically facilitate cell proliferation and viability [53].

4. Cav-1 Participates in Lung Cancer Metastasis

4.1. Cav-1 and Angiogenesis

Like other cancers, lung cancer is composed of parenchyma and stroma. Parenchyma is the real tumor mass that consists of true neoplastic cells. The major components of stroma are connective tissues and blood vessels. They can support and nourish the parenchymal portion. To meet the nutritional requirements, new blood vessels in lung cancer are constantly formed based on the existing networks in the stroma, which can also provide pathways for cancer cell hematogenous migration [58,59,60]. In addition, the vasculature in cancers is more permeable than that in normal tissues, therefore making it easier for cancer cells to migrate [61]. Vascular endothelial growth factor (VEGF) family proteins can be secreted by tumor cells. They can bind to the VEGF receptor (VEGFR) on the endothelial cell membrane to regulate endothelial cell proliferation and migration, which is regarded as angiogenesis [62,63,64].

Some studies have demonstrated the links between Cav-1 and the angiogenesis that occurs in lung cancer. In Lewis lung carcinoma (LLC) cells (NSCLC cell line), cavtratin, the scaffolding domain peptide of Cav-1, can inhibit endothelial nitric oxide synthase (eNOS) function and subsequently block NO production, eventually to prevent LLC cells leakage and decrease angiogenesis [61,65,66,67]. In Cav-1-knockout mice transplanted with LLC cells, the Cav-1 absence prevents VEGFR-2 from binding VE-cadherin (a protein that can interact with VEGFR-2 and lead to its dephosphorylation) and permits VEGFR-2 to be phosphorylated by other factors such as VEGF families, thus to enhance endothelial cell growth and lead to angiogenesis. Such mice also show microvascular hyperpermeability [68,69]. These findings imply that Cav-1 can negatively regulate lung cancer angiogenesis and prevent tumor cell leakage.

4.2. Cav-1 and Pseudopods Formation

Pseudopods, including filopodia, lamellipodia and invadopodia, are cellular protrusions that can mediate cell motility and sensation [54]. Filopodia and lamellipodia have similar functions but are structurally different. Filopodia are finger-like protrusions while lamellipodia are flatter and broader, both of which are important for cell migration [70]. There are complex and massive molecules associated with pseudopods formation. FAK is a non-receptor tyrosine kinase that is up-regulated in various cancers, and involved in tumor cell proliferation, migration and survival. By recruiting the downstream molecules paxillin and talin, FAK is able to modulate focal adhesion dynamics to control cell motility [71,72]. It can also interact with the p85 subunit of phosphatidylinositol 3-kinase (PI3K) to phosphorylate AKT [73,74]. AKT can mediate actin organization by acting on Girdin, an actin-binding protein [75,76]. Actin polymerization and rearrangement eventually trigger the generation of pseudopods [77,78].

Cav-1 can mainly induce the formation of filopodia and lamellipodia, thus enhancing NSCLC cell migration. In NO-treated H460 cells, Cav-1 expression is upregulated, which subsequently elevates phosphorylated FAK (pFAK) and phosphorylated AKT (pAKT) levels. As a result, such cells generate more filopodia and show stronger motility and invasiveness [28]. In H23 cells, with a fluorescence microscope, the lamellipodia number is observed to be increased due to Cav-1 over-expression, which can elevate pAKT expression leading to enhanced cell invasiveness [79]. Similar to the aforementioned results, Ho et al. also reported that in the highly-invasive lung AC cell lines (CL1-5 and CL1-5F4), Cav-1 expressed higher than that in the low-invasive cell lines (CL1-0 and CL1-1). Such high expression of Cav-1 induced the formation of filopodia and eventually enhanced cell motility. Additionally, they demonstrated that the lipid existence was indispensable for Cav-1-mediated filopodia formation [80]. On the contrary, in H460 and H292 cells, chrysotobibenzyl-induced Cav-1 downregulation leads to decreases in pFAK and pAKT, which suppresses filopodia generation and cell migration [81].

4.3. Cav-1 and EMT

During EMT, cells lose their original characteristics and gain mesenchymal properties. The original cobblestone-like epithelial cells are converted to spindle-like mesenchymal cells. The expression of epithelial markers, such as E-cadherin, are downregulated, while mesenchymal markers such as N-cadherin, Vimentin, Snail, Slug, Twist and alpha-smooth muscle actin (α-SMA) are over-expressed. Such cells can also secrete MMPs to degrade the basement membrane, thus permitting the cells to invade and migrate [82,83,84,85].

In NSCLC cell lines, Cav-1 can exert dual roles on EMT progression. In A549 cells, transforming growth factor-β (TGF-β) can bind to the TGF-β receptor-1 (TβR-1) on the cell membrane, which triggers EMT by upregulating Vimentin, Snail and Slug, and downregulating E-cadherin. Cav-1 can also combine with TβR-1 to induce its internalization and further cause its degradation mediated by ubiquitin-proteosome, thus blocking TGF-β signaling pathway. In contrast, syntenin, a scaffold protein that can interact with multiple membrane receptors, is able to prevent TβR-1 internalization and degradation by disassociating the TβR-1/Cav-1 complex, finally to facilitate lung cancer progression [86,87]. A metastasis-associated protein called EF-hand domain-containing protein D2 (EFHD2) is found to inhibit Cav-1 mRNA and protein expressions, and subsequently upregulate Snail and Vimentin as well as downregulate E-cadherin in A549 cells to enhance EMT. Additionally, Cav-1 re-expression can partly reverse EFHD2-induced EMT [88]. E-cadherin can link to β-catenin on the cell membrane and then strengthen cell adhesion. In H460 cells, Cav-1 knockdown can reduce E-cadherin and increase β-catenin, hence, attenuating cell adhesion leading to EMT process [48,89]. The aforementioned studies suggest an inhibitory role of Cav-1 in regulating EMT in NSCLC. However, in some occasions, Cav-1 may function as a positive regulator. In H157 cells, Cav-1 over-expression can increase the Snail level, which further represses the E-cadherin level to facilitate EMT and migration [90,91]. Conversely, in Cav-1-knockdown H460 and 95D cells, decreased pEGFR, pERK1/2, MMP-2 and MMP-9 levels can be measured by western blot and reduced cell invasion can be observed by transwell assay [92,93]. In Cav-1 over-expressed SCLC cell line H446, EMT-associated characteristics such as low E-cadherin expression and high MMP-3 expression can be detected, resulting in enhanced cell invasion [94].

4.4. Cav-1 Regulates Cell Movement via Multiple Molecules

There are some other pathways involved in Cav-1-mediated NSCLC cell motility. Located on the cell membrane, caveolae can sense the alteration of mechanical stress, and Cav-1 can be phosphorylated responding to several stimuli, thus, to be involved in multiple cellular events [6]. Sinha et al. found that mechanical stress over Hela and mouse lung endothelial cells flattened caveolae to buffer the tension and freed Cav-1 [95]. In A549 and CL1-5 cells, elevated hydrostatic pressure (which mimics increased interstitial fluid pressure that is seen in many solid cancers) can increase phosphorylated Cav-1. Then, through Cav-1/AKT/ERK axis, the aquaporin-1 (AQP1) level is elevated, which can impel cell migration [96]. In A549 cells, Cav-1 and protein arginine methyltransferase 5 (PRMT5) can synergistically facilitate the exteriorization of Eno-1, a glycolytic enzyme that can promote lung cancer cell migration. Eno-1 is further methylated by PRMT5 and then degrades the ECM for cell invasion [97]. Hydrogen peroxide (H₂O₂) and hydroxyl radical (·OH) are two key exogenous reactive oxygen species (ROS). In H460 cells, Cav-1 over-expression can elevate pAKT expression and subsequently suppress the production of H₂O₂ and ·OH. The dysregulation of ROS negatively influences the expression of vascular endothelial cell adhesion molecule-1 (VCAM-1). VCAM-1 can promote the adhesion between endothelium and cancer cells. Hence, its ablation makes such an attachment become loose, leading to promoted cell migration and invasion [98]. H₂O₂ and another ROS superoxide anion (O₂^·−) can decrease the Cav-1 level by enhancing its ubiquitination in H460 cells. Cav-1 downregulation then decreases pAKT, and results in attenuated cell migration and invasion. Nevertheless, ·OH treatment can produce entirely opposite results in comparison with the other two ROS [99]. Cav-1 can also function to inhibit lung cancer cell migration. In H1299 cells, Cav-1 can interact with DLC-1, an anti-metastasis protein, to co-localize in caveolae and form a complex. It can inhibit tumor cell migration probably by enhancing the tumor suppressive role of Cav-1 in these cells [100].

5. Cav-1 Involved in Lung Cancer Therapy

Current treatments of lung cancer include surgery, chemotherapy, radiotherapy and targeted therapy. The traditional surgical resection of the tumor is generally recommended for early-stage lung cancer (especially NSCLC) patients [125]. First-line drugs containing platinum (e.g., cisplatin and carboplatin) plus paclitaxel, and other chemotherapeutic agents such as gemcitabine, vinblastine and docetaxel are routinely applied for NSCLC chemotherapy [126,127]. Targeted drugs aimed at EGFR mutation, regarded as EGFR tyrosine kinase inhibitors (EGFR-TKI), are also well studied [128]. However, a major obstacle called multiple drug resistance (MDR) can usually affect drug efficacy when using these drugs as treatment.

5.1. Cav-1 and Drug Resistance

A high Cav-1 level has been shown to be associated with drug resistance during the treatment process of numerous cancers such as lung cancer, esophageal cancer [129], colorectal cancer [130] and renal carcinoma [131]. Cav-1 can enhance drug resistance in some NSCLC cell lines. In the parental paclitaxel-sensitive A549 cells, caveolae are absent. However, both caveolae and Cav-1 are upregulated in paclitaxel-resistant A549 cells, which implies a potential relationship between Cav-1 and paclitaxel resistance [132]. Conversely, Cav-1 knockdown in such paclitaxel-resistant A549 cells can induce apoptosis by increasing Bax and decreasing Bcl-2, thus, making the cells become sensitive to paclitaxel [49]. In PC-9 cells treated by gefitinib and erlotinib separately, Cav-1 knockdown results in attenuated cell proliferation and migration, and they become more sensitive to gefitinib and erlotinib. These are probably the consequences of Cav-1 downregulation-mediated decreases of pEGFR, pERK and pAKT. The same results were also observed in tumor-implanted nude mice treated exclusively with gefitinib [133]. In H292 cells, NO exposure can upregulate Cav-1, pAKT and Bcl-2, finally trigger chemo-resistance against doxorubicin and etoposide as well as promote cell survival, which were assessed by Hoechst 33342 staining and flow cytometry [134].

Unlike the aforementioned observations, some papers have also reported that Cav-1 can negatively regulate drug resistance in NSCLC cell lines. A high Cav-1 level could contribute to the entry of albumin and nab-paclitaxel (albumin-bound paclitaxel) into H23 lung cancer cells, therefore, enhancing cell apoptosis; while drug resistance is shown in these cells following Cav-1 reduction [135]. According to such a mechanism, a novel approach has been designed to maximize the efficacy of fenretinide. In A549 cells, Cav-1 upregulation can efficiently increase the absorption of fenretinide encapsulated by albumin shells, thus promoting fenretinide-induced toxicity in A549 cells [136]. P-glycoprotein (P-gp) can transport drugs from the cytosol to the matrix. Over-expression of P-gp can promote drug efflux, lower the intracellular drug concentration, reduce drug’s cytotoxicity, hence, leading to drug resistance [137,138]. It has been reported that P-gp is found highly expressed in caveolae-rich areas and can interact with Cav-1. Such an interaction can inhibit P-gp-mediated drug efflux to decrease drug resistance. For example, in doxorubicin-resistant Hs578T breast cancer cells, Cav-1 over-expression can reduce the activity of P-gp and corresponding doxorubicin resistance [139,140]. When the combination of Cav-1 with P-gp is interrupted, the function of P-gp can be reactivated. In A549 and H460 cells, Cav-1 with lysine 176-to-arginine (K176R) mutant fails to form the Cav-1/P-gp complex, thus P-gp functions normally to accelerate the efflux of substrates (i.e., paclitaxel and doxorubicin). Moreover, they also found that Cav-1 K176R mutant could influence Cav-1 oligomerization, which is essential for caveolae structure and is probably the cause for inducing the disassociation of Cav-1 and P-gp [141].

5.2. Cav-1 and Cell Senescence

Cell senescence refers to a physiologically inactive state and such cells undergo irreversible growth arrest [142]. Therefore, cell senescence can be regarded as a novel method of interrupting cancer cell proliferation and treating cancers [143]. In general, bleomycin can modulate Cav-1 expression, give rise to cell senescence and act as an anti-tumor agent [144]. In A549 cells, bleomycin treatment can elevate the Cav-1 level and then downstream molecules p53 and p21 [145]. Additionally, the Cav-1-induced activation of the p53/p21 pathway has been previously demonstrated to inhibit the function of CDK and inactivate DNA polymerase δ (a key regulator of DNA replication) to cause cell cycle arrest in NIH3T3 fibroblast cells [146]. Additionally, Cav-1 is also found to interact with MGr1-Ag on plasma membrane in the bleomycin-treated A549 cells. Their binding can potentially lead to cell senescence, however, further work is still required [147].

5.3. Multiple Cav-1-Targeted Agents

In the previous context, it has been discussed that multiple endogenous and exogenous agents can modulate Cav-1 expression to regulate lung cancer progression. There are also several natural bioactive compounds that can function similarly. They all target Cav-1 to inhibit NSCLC progression (Table 2). Some of them can inhibit lung cancer cell migration and invasion, such as chrysotobibenzyl and gigantol. Chrysotobibenzyl, a compound isolated from Dendrobium pulchellum, can suppress Cav-1 expression and then lead to the downregulation of integrin β1, β3, αv and downstream effectors pFAK and pAKT, thus inhibiting H292 and H460 cell migration [81]. In H460 cells, gigantol, a bibenzyl compound from Dendrobium draconis, can cause a decrease in Cav-1 levels and then pAKT to subsequently suppress Cdc42 expression and filopodia formation, finally to attenuate cell motility [148]. Others can elevate lung cancer cells' apoptotic rates, including dendrofalconerol A (DF-A), moscatilin, zinc, cordycepin, jorunnamycin A and ethanolic extract of Antrodia cinnamomea (EEAC). DF-A, a methanol extracted from Dendrobium falconeri, can elevate the sensitivity of H460 cells to anoikis by means of decreasing Cav-1, pAKT and Bcl-2 levels [113]. Moscatilin, isolated from Dendrobium brymerianum, can also induce anoikis by reducing pro-survival proteins pATK, pERK and Mcl-1 via a Cav-1-dependent pathway in H460 cells [149]. Zinc can sensitize H460 cells to anoikis by suppressing Cav-1 and pAKT [150]. In A549 cells, cordycepin, an adenosine analog, can raise Cav-1 expression. Cav-1 then elevates phosphorylated JNK (p-JNK) that can further prevent Foxo3a phosphorylation and enhance its nuclear translocation. In the nucleus, Foxo3a can increase the expression of Bax and caspase-3, then the cells exhibit reinforced apoptosis [151]. Jorunnamycin A is an extract from Xestospongia sp., which can suppress Cav-1 expression to inhibit EMT and survival in H460, H292 and H23 cells [152]. By decreasing Cav-1 expression, EEAC can make A549 cells more sensitive to paclitaxel treatment and slow down cell viability [153].

Table 2. Various Cav-1-targeted approaches to suppressing NSCLC progression.

Agent	Cell Line	Mode of Action	Reference
H2O2 and O2·−	H460	H2O2 and O2·−→ Cav-1↓→ pAKT↓→ migration↓	[99]
Chrysotobibenzyl	H460 H292	Chrysotobibenzyl→ Cav-1↓→ integrin β1, β3, αv↓ → pFAK↓ and pAKT↓→ migration↓	[81]
Gigantol	H460	Gigantol→ Cav-1↓→ pAKT↓→ CDC42↓→ EMT↓ and migration↓	[148]
DF-A	H460	DF-A→ Cav-1↓, Mcl-1↓and Bcl-2↓→ anoikis↑	[114]
Moscatilin	H460	Moscatilin→ Cav-1↓→ Mcl-1↓→ pAKT↓,pERK↓→ anoikis↑	[149]
Zinc	H460	Zinc→ Cav-1↓→ pAKT↓→ anoikis↑	[150]
Cordycepin	A549	Cordycepin→ Cav-1↑→ p-JNK↑→ Foxo3a↑→ Bax↑→ apoptosis↑	[151]
Jorunnamycin A	H460 H292 H23	Jorunnamycin A→ Cav-1↓→ pAKT↓,pERK↓→ EMT↓ and apoptosis↑	[152]
EEAC	A549	EEAC→ Cav-1↓→ chemosensitivity to paclitaxel↑	[153]
Albumin-encapsuled fenretinide	A549	Cav-1 promotes albumin-encapsuled fenretinide uptake into cell→ apoptosis↑	[136]
Bleomycin	A549	Bleomycin→ Cav-1↑→ p53↑, p21↑→ senescence↑	[145]