Anoikis-Associated Lung Cancer Metastasis: Mechanisms and Therapies

Simple Summary Anoikis is a programmed cell death process resulting from the loss of interaction between cells and the extracellular matrix. Therefore, it is necessary to overcome anoikis when tumor cells acquire metastatic potential. In lung cancer, the composition of the extracellular matrix, cell adhesion-related membrane proteins, cytoskeletal regulators, and epithelial–mesenchymal transition are involved in the process of anoikis, and the initiation of apoptosis signals is a critical step in anoikis. Inversely, activation of growth signals counteracts anoikis. This review summarizes the regulators of lung cancer-related anoikis and explores potential drug applications targeting anoikis. Abstract Tumor metastasis occurs in lung cancer, resulting in tumor progression and therapy failure. Anoikis is a mechanism of apoptosis that combats tumor metastasis; it inhibits the escape of tumor cells from the native extracellular matrix to other organs. Deciphering the regulators and mechanisms of anoikis in cancer metastasis is urgently needed to treat lung cancer. Several natural and synthetic products exhibit the pro-anoikis potential in lung cancer cells and in vivo models. These products include artonin E, imperatorin, oroxylin A, lupalbigenin, sulforaphane, renieramycin M, avicequinone B, and carbenoxolone. This review summarizes the current understanding of the molecular mechanisms of anoikis regulation and relevant regulators involved in lung cancer metastasis and discusses the therapeutic potential of targeting anoikis in the treatment of lung cancer metastasis.


Introduction
Lung cancer is one of the most malignant tumors worldwide, characterized by high morbidity and mortality [1]. The outcomes for patients with metastatic lung cancer are poor, with a 5-year survival rate of <5% [2]. Tumor metastasis is a complex multistep process, including migration from cancer tissue, intravasation, survival in the circulatory system, extravasation, homing, and metastatic colonization [3][4][5]. To finish the initial migration step, tumor cells must interrupt cell-cell and cell-stromal interactions, become

ECM and Cell Adhesion
3.1.1. ECM ECM comprises fibronectin, laminins, collagens, elastin, and several other glycoproteins. They bind with cell adhesion receptors to form a three-dimensional macromolecular network, which regulates various cell functions, including survival, growth, morphology, and migration [15]. Tumor cells undergo anoikis due to loss or inappropriate cell adhesion to ECM [16]. Acquisition of anchorage-independent survival requires tumor cells survival when detached from the ECM matrix [17,18]. In different artificially mimicked ECM compositions, A549 cells exhibited different sensitivity to doxorubicin and anoikis in vitro. Downregulation of focal adhesion kinase (FAK) signaling is accompanied by anoikis sensitization [18]. As for ECM components, fibronectin is upregulated for cell aggregate formation during cell detachment, which enhances anoikis resistance in lung cancer. Fibronectin knockdown decreases glycoproteins, including desmoglein-2, desmocollin-2/3, and plakoglobin, to increase anoikis [19]. Laminin 5 expression is significantly correlated with advancement along the alveolar wall growth pattern and metastasis in lung adenocarcinoma. Laminin 5 activates integrin/FAK signaling to induce anoikis resistance in several lung adenocarcinoma cell lines suspended in soft agar-coated dishes [20]. Liu et al. showed that collagen XVII cooperated with laminin 5 to activate FAK and mediate suspension survival. The authors found that activation of PP2A/STAT3 promotes collagen XVII-induced suspension survival, which determined anoikis resistance and initial metastasis in A549 cells and malignant lung cancer pleural effusion mouse models [21]. Highly expressed collagen IV facilitated liver metastasis in lung cancer patients. Burnier et al. investigated lung-metastasizing M-27 cells and found that collagen IV silencing enhanced anoikis through the integrin α2/FAK axis in vitro and reduced liver metastasis by inoculation of tumor cells into the intrasplenic/portal system in vivo [22]. These findings suggest that upregulation of ECM components can mediate cell survival and re-adhesion 3.1. ECM and Cell Adhesion 3.1.1. ECM ECM comprises fibronectin, laminins, collagens, elastin, and several other glycoproteins. They bind with cell adhesion receptors to form a three-dimensional macromolecular network, which regulates various cell functions, including survival, growth, morphology, and migration [15]. Tumor cells undergo anoikis due to loss or inappropriate cell adhesion to ECM [16]. Acquisition of anchorage-independent survival requires tumor cells survival when detached from the ECM matrix [17,18]. In different artificially mimicked ECM compositions, A549 cells exhibited different sensitivity to doxorubicin and anoikis in vitro. Downregulation of focal adhesion kinase (FAK) signaling is accompanied by anoikis sensitization [18]. As for ECM components, fibronectin is upregulated for cell aggregate formation during cell detachment, which enhances anoikis resistance in lung cancer. Fibronectin knockdown decreases glycoproteins, including desmoglein-2, desmocollin-2/3, and plakoglobin, to increase anoikis [19]. Laminin 5 expression is significantly correlated with advancement along the alveolar wall growth pattern and metastasis in lung adenocarcinoma. Laminin 5 activates integrin/FAK signaling to induce anoikis resistance in several lung adenocarcinoma cell lines suspended in soft agar-coated dishes [20]. Liu et al. showed that collagen XVII cooperated with laminin 5 to activate FAK and mediate suspension survival. The authors found that activation of PP2A/STAT3 promotes collagen XVII-induced suspension survival, which determined anoikis resistance and initial metastasis in A549 cells and malignant lung cancer pleural effusion mouse models [21]. Highly expressed collagen IV facilitated liver metastasis in lung cancer patients. Burnier et al. investigated lung-metastasizing M-27 cells and found that collagen IV silencing enhanced anoikis through the integrin α2/FAK axis in vitro and reduced liver metastasis by inoculation of tumor cells into the intrasplenic/portal system in vivo [22]. These findings suggest that upregulation of ECM components can mediate cell survival and re-adhesion to promote metastasis of tumor cells, primarily through integrin/FAK. A summary is presented in Figure 2. to promote metastasis of tumor cells, primarily through integrin/FAK. A summary is presented in Figure 2.

Integrins
Integrins are a family of cell surface heterodimeric receptors consisting of noncovalently linked α-and β-subunits that determine the receptor affinity for ECM. Integrinmediated tumor stroma sensing, stiffening, and remodeling are critical steps in cancer progression that support tumor invasion, acquiring tumor stem cell properties, and drug resistance [23]. Integrins help metastatic cancer cells facilitate anchorage-independent survival and resist anoikis [24,25], and abnormal integrin expression contributes to several cancers' metastasis [26]. In addition, integrin signaling was stimulated by growth factors to induce crosstalk. Integrin β3 was activated by transforming growth factor-β1 (TGF-β1) to increase the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor resistance, then EMT and anoikis resistance was observed in lung cancer cell lines [27]. Several studies have proven that FAK was a major downstream effector resisting anoikis through the Src or ERK pathway [20,22,25]. Furthermore, membrane proteins carcinoembryonic antigen (CEA) and cellular retinoic acid binding protein 2 (CRABP2) engage in integrin signaling to suppress anoikis [28]. Wu et al. found that CRABP2 promoted integrin β1/FAK/ERK signaling and inhibited anoikis. Overexpressed CRABP2 increased lymph node and liver metastasis through in vivo tail vein injection of lung cancer cell models [25].

Integrins
Integrins are a family of cell surface heterodimeric receptors consisting of noncovalently linked αand β-subunits that determine the receptor affinity for ECM. Integrinmediated tumor stroma sensing, stiffening, and remodeling are critical steps in cancer progression that support tumor invasion, acquiring tumor stem cell properties, and drug resistance [23]. Integrins help metastatic cancer cells facilitate anchorage-independent survival and resist anoikis [24,25], and abnormal integrin expression contributes to several cancers' metastasis [26]. In addition, integrin signaling was stimulated by growth factors to induce crosstalk. Integrin β3 was activated by transforming growth factor-β1 (TGF-β1) to increase the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor resistance, then EMT and anoikis resistance was observed in lung cancer cell lines [27]. Several studies have proven that FAK was a major downstream effector resisting anoikis through the Src or ERK pathway [20,22,25]. Furthermore, membrane proteins carcinoembryonic antigen (CEA) and cellular retinoic acid binding protein 2 (CRABP2) engage in integrin signaling to suppress anoikis [28]. Wu et al. found that CRABP2 promoted integrin β1/FAK/ERK signaling and inhibited anoikis. Overexpressed CRABP2 increased lymph node and liver metastasis through in vivo tail vein injection of lung cancer cell models [25].

CEA
CEA family members are involved in the biological function of intercellular adhesion [29]. First, Camacho-Leal et al. found that the expression of CEA is closely related to integrins, and deleting the CEA functional domain weakened the cross-linking of integrin α5β1 and cell adhesion to fibronectin, which led to anoikis [28]. Subsequently, antibodymediated CEA deletion attenuated specific binding to integrin α5β1. The co-clustered CEA and integrin α5β1 activated integrin-linked kinase (ILK), AKT, and ERK/mitogen-activated protein kinase (MAPK) pathways to impair anoikis [30]. Furthermore, CEA reduces anoikis through the downregulation of the intrinsic cell death pathway, and the inactivation of caspase-9 and -3 [31]. The above studies demonstrate the effect of CEA on anoikis resistance. In addition, CEA-related cell adhesion molecule 6 (CEACAM6) is upregulated in lung adenocarcinoma patients with poor outcomes. Homophilic interactions of CEACAM6 between lung cancer cells and the tumor microenvironment could inhibit anoikis through Src/FAK pathway activation [32].

CUB-Domain-Containing Protein 1 (CDCP1)
CDCP1 is an Src family kinase-binding phosphoprotein implicated in promoting metastasis via anoikis inhibition [36,37]. Phosphorylation of CDCP1 enhanced anchorageindependent growth of A549, PC14, H520, and H322 in a colony formation assay on soft agar. A549 cells with knockdown CDCP1 injected into the tail veins of a mouse model were found to have fewer metastatic nodules [36]. Regarding the mechanism of CDCP1 against anoikis, a few studies have proven that Ras activated CDCP1; subsequently, Tyr734 of CDCP1 bound to Fyn and was phosphorylated to activate protein kinase Cδ (PKCδ) and inhibit autophagy, helping tumor cells to escape apoptosis [36,38,39].

FAK
FAK is a ubiquitously expressed non-receptor tyrosine kinase that regulates cellular functions from embryonic development to wound healing, cell adhesion, cell migration, and angiogenesis [40,41]. For cell adhesion, FAK is recruited by integrins that form a dual kinase complex. The complex responds to signals from ECM components, including fibronectin and collagen [20,22,25]. FAK modulates both cell death-and survival-related pathways to resist anoikis. On the one hand, FAK binds to Src or directly activates the phosphatidylinositide-3-kinase (PI3K)/AKT, ERK/MAPK, and p38/MAPK pathways for survival during cell detachment [22,25,42]. On the other hand, FAK upregulates the phosphorylation of p190RhoGAP and inhibits RhoA-triggered pro-apoptotic phosphorylation of BCL-2-interacting mediator of cell death (BIM) to escape from anoikis [10]. Olfactomedin III is highly expressed in anoikis-resistant lung cancer cell lines and promotes the phosphorylation of FAK to keep procaspase-3 from activation [43]. Conversely, apoptosis signals inhibit FAK. X-linked ectodermal dysplasia receptor (XEDAR) was an essential effector downstream of p53 and negatively regulated FAK to induce anoikis [44].

Tyrosine-Protein Kinase Src
Cellular functions of Src kinases are involved in the cell cycle, proliferation, differentiation, adhesion, and angiogenesis [45]. These functions are mediated by Src kinases acting as membrane adhesion molecular switches, linking various extracellular signals to intracellular signaling pathways [46]. Src phosphorylation creates anoikis resistance and causes lung cancer cells to "float" in lymph nodes [47]. Src is a membrane-attached molecule regulated by several pathways to attenuate anoikis. FAK recruits Src to inhibit anoikis as previously mentioned [42]. It is also reported that Pyk2 appeared to be the critical downstream effector of Src and induced metastasis in lung cells [48]. Furthermore, Wei et al. found that Src Tyr418 phosphorylation triggered phosphorylation of p130 Cas and caused anchorage-independent growth and metastasis in lung cancer [49].
3.1.8. p66 Shc p66 Shc is an SHC adapter protein 1 (SHC1) gene-encoding protein, localized to focal adhesions to permit anchorage-dependent growth and promote anoikis function as a tumor metastasis suppressor [53]. Expression of p66 Shc in lung cancer samples correlated with good outcomes and anoikis [54]. p66 Shc promotes anoikis in lung cancer through the perception of cell attachment, inhibition of proliferation, and promotion of apoptosis. p66 Shc restrains Ras and Rac1 hyperactivation and increases RhoA activation to require focal adhesion and restore anoikis [55,56]. Typically, p66 Shc did not affect cell death in adherent cells. During cell detachment, p66 Shc translocated into mitochondria and bound to cytochrome c to release and cause death [55]. On the other hand, in the absence of adhesion conditions, p66 Shc induced autophagy and activated apoptosis-associated protein cleavage of caspase-7 and poly (ADP-ribose) polymerase (PARP) by inhibiting phosphorylation of ERK1/2 [57,58]. DNA methylation in promoter-mediated epigenetic repression of p66 Shc increased cancer cell survival and tumor progression [59]. Aiolos is an Ikaros zinc finger family member associated with epigenetic modifications [60]. Aiolos acts as an oncogene to disrupt enhancer-promoter interactions of p66 Shc to inhibit transcription [61]. Aiolos also silences the zinc finger transcription factor PR domain containing 1 (PRDM1), resulting in anoikis resistance [62]. Similarly, zinc-finger E-box binding protein 1 (ZEB1) repressed the p66 Shc promoter. Furthermore, ZEB1 potentiates EMT and blocks anoikis by increasing vimentin and decreasing E-cadherin and β-catenin [63].

EGFR
EGFR is a transmembrane protein that transduces intracellular growth factor signals, promotes tumor proliferation and metastasis, and is an essential target of current lung cancer therapy. In the absence of cell adhesion, normal cells lose expression of EGFR and induce apoptosis; by contrast, tumor cells do not exhibit loss of EGFR during detachment [64]. In growth signaling, EGFR acquires survival and anoikis resistance by activating Ras, ERK, and PI3K/AKT pathways [51,65,66]. EGFR has crosstalk through cell-adhering proteins or inhibits apoptosis signaling to inhibit anoikis. EGFR and integrins cooperatively inhibited anoikis by downregulating the BIM during cell detachment from the ECM [27,64]. NADPH oxidase 4 (NOX4) is a reactive oxygen species (ROS)-generating enzyme. NOX4 increased the activation of EGFR through ROS generation; a soft agar colony assay showed that si-NOX4 and si-EGFR attenuated Src expression and enhanced anoikis [51]. Regulators that mediate EGFR protein degradation are also involved in the regulation of anoikis. E3-ubiquitin ligase c-Cbl is an essential ligand for EGFR degradation. CCN family protein 2 (CCN2) bound EGFR and recruited E3-ubiquitin ligase for EGFR ubiquitination and degradation [66]. FAM188B prevented EGFR from degrading to cause lung cancer cells to re-adhere to the ECM [52].

Neurotrophic Tyrosine Kinase Receptor 2 (NTRK2/TrkB)
TrkB is a neurotrophin that serves as a growth factor regulating embryonic stem cells and promoting tumorigenesis in cancer cells. TrkB is upregulated in anoikis-resistant lungderived tumor cells isolated from malignant ascites [67]. TrkB activates EMT, tumorigenesis, and lung metastasis and attenuates anoikis via the twist/snail axis in breast and lung cancer [68]. The expression of TrkB and E-cadherin had an inverse relationship in a panel of lung adenocarcinoma samples [69]. Cancer-derived TrkB mutations partly altered the functional characterization of the protein. TrkB T695I and TrkB D751N in colon cancer-derived mutants showed less activity than wild-type TrkB, and the function of TrkB L138F in lung cancer and TrkB P507L in breast cancer was indistinguishable from wild-type [70].

βIII-Tubulin
βIII-tubulin is a microtubule protein constituent of the cytoskeleton. It contributes to tumor metastasis and chemotherapy resistance [82]. βIII-tubulin contributed to anti-anoikis and metastasis in lung cancer. High levels of βIII-tubulin inhibited the phosphatase and tensin homolog deleted on chromosome ten (PTEN) and enhanced phosphorylation of AKT to induce tumor spheroid outgrowth and anoikis resistance in NSCLC cells [76].

βIII-Tubulin
βIII-tubulin is a microtubule protein constituent of the cytoskeleton. It contributes to tumor metastasis and chemotherapy resistance [82]. βIII-tubulin contributed to anti-anoikis and metastasis in lung cancer. High levels of βIII-tubulin inhibited the phosphatase and tensin homolog deleted on chromosome ten (PTEN) and enhanced phosphorylation of AKT to induce tumor spheroid outgrowth and anoikis resistance in NSCLC cells [76].

Rho and Rho-Associated Kinase (ROCK)
The Rho family of GTPases are small signaling G proteins from the Ras superfamily. RhoA regulates cell morphology change, cell-matrix adhesion, and cytoskeletal reorganization [83,84]. Cell adhesion molecules regulate RhoA; activation of FAK counteracted RhoA inducing anoikis [10], and p66 Shc promoted RhoA, causing anoikis [56]. RhoB can inhibit invasion and proliferation and induce anoikis, whereas growth signals Ras, ERK, and PI3K/AKT counteract the effects of RhoB [65,73]. In summary, RhoA and RhoB promote anoikis and antagonize Ras. ROCK kinases are critical downstream effectors of Rho GTPases [85]. Haun et al. showed that RhoA/ROCK activated MKK4/MKK7 and JNK to promote BIM phosphorylation and caspase-3 processing, activating the anoikis signaling pathway [10]. A summary of cytoskeleton and regulators is presented in Figure 4.

Rho and Rho-Associated Kinase (ROCK)
The Rho family of GTPases are small signaling G proteins from the Ras superfamily. RhoA regulates cell morphology change, cell-matrix adhesion, and cytoskeletal reorganization [83,84]. Cell adhesion molecules regulate RhoA; activation of FAK counteracted RhoA inducing anoikis [10], and p66 Shc promoted RhoA, causing anoikis [56]. RhoB can inhibit invasion and proliferation and induce anoikis, whereas growth signals Ras, ERK, and PI3K/AKT counteract the effects of RhoB [65,73]. In summary, RhoA and RhoB promote anoikis and antagonize Ras. ROCK kinases are critical downstream effectors of Rho GTPases [85]. Haun et al. showed that RhoA/ROCK activated MKK4/MKK7 and JNK to promote BIM phosphorylation and caspase-3 processing, activating the anoikis signaling pathway [10]. A summary of cytoskeleton and regulators is presented in Figure 4.

Cell Detachment and Directional Migration
CAV1 is a vital component of plasma membrane caveolae; it undergoes extracellular changes and regulates caveola-dependent signaling and endocytosis [86]. CAV1 is a cellular membrane raft structure that responds to external environmental stimuli such as ROS, shear stress, and mechanical stress. In addition, CAV1 can regulate cell polarization and directional migration [87]. CAV1 functions as a membrane adaptor to kinase Fyn in integrin signaling, critical for anchorage-dependent cell growth [88]. Several studies have proven that CAV1 overexpression enhanced anchorage-independent growth in H460 cells in vitro [89,90]. Myeloid leukemia-1 (MCL-1) is an anti-apoptotic protein required to escape anoikis in several tumors, including breast cancer, osteosarcoma, and melanoma [91,92]. CAV1 interacted with MCL-1 to prevent degradation and resist anoikis [93].

Cell Detachment and Directional Migration
CAV1 is a vital component of plasma membrane caveolae; it undergoes extracellular changes and regulates caveola-dependent signaling and endocytosis [86]. CAV1 is a cellular membrane raft structure that responds to external environmental stimuli such as ROS, shear stress, and mechanical stress. In addition, CAV1 can regulate cell polarization and directional migration [87]. CAV1 functions as a membrane adaptor to kinase Fyn in integrin signaling, critical for anchorage-dependent cell growth [88]. Several studies have proven that CAV1 overexpression enhanced anchorage-independent growth in H460 cells in vitro [89,90]. Myeloid leukemia-1 (MCL-1) is an anti-apoptotic protein required to escape anoikis in several tumors, including breast cancer, osteosarcoma, and melanoma [91,92]. CAV1 interacted with MCL-1 to prevent degradation and resist anoikis [93].
Tumor cells suffer fluid shear stress through the venous and lymphatic systems, which can acquire anoikis resistance. CAV1 can help cells overcome fluid shear stress and anoikis [94,95]. Free radicals stimulate CAV1 to a large extent during cell detachment. Oxidative stress products, including nitric oxide (NO) and hydrogen peroxide (H2O2), inhibited CAV1 degradation to resist anoikis [90,96]. NO-mediated S-nitrosylation of CAV1 is a protein modification that stabilizes CAV1 protein and protects it from ubiquitination [89]. As feedback, anoikis resistance enhanced CAV1 expression [97,98]. H2O2 inhibited the formation of the CAV1-ubiquitin complex through the consumption of catalase and N-acetylcysteine to cause anoikis resistance in H460 cells [96,[99][100][101].

EMT
EMT is characterized by epithelial cells undergoing remarkable morphologic changes to the mesenchymal phenotype, leading to increased motility and invasion in cancer [104]. Loss of the epithelial marker E-cadherin makes cells lose attachment and antagonizes the response to anoikis after cell detachment [105,106]. Some features of EMT, including loss of the epithelial cell-cell junction, reorganization of the actin cytoskeleton, and gain of directional migration capability, are critical for anoikis resistance [107,108]. Upregulation of N-cadherin, vimentin, twist, snail, and ZEB1, feature proteins of EMT, inhibits anoikis [63,68,109]. In summary, EMT is a process of adaptation to anchorage-independent growth, which is a factor against anoikis. Some critical proteins (i.e., integrin, claudin-1, p66 Shc , RhoA, CAV1, contactin 1, and TrkB) are involved in EMT in the context of anoikis, as previously mentioned [63] [27,33,68]. RhoA downstream signaling can affect actin remodeling and prevent the formation of contractile stress fibers during EMT [109]. Several pathways (i.e., the PI3K/AKT, Notch, and Wnt pathways) are related to EMT. Contactin 1 promotes EMT and anoikis resistance by enhancing the PI3K/AKT pathway [106]. Notch-1 enhanced the expression of vimentin and snail to suppress anoikis [110]. Fas apoptotic inhibitory molecule 2 (FAIM2) induced EMT and anoikis resistance through the Wnt/β-catenin pathway in NSCLC bone metastasis [111]. NO exposure upregulated EMT and enhanced anoikis resistance, as shown by anoikis assays with poly-HEMA-coated plates [90]. DAPK is a Ser/Thr protein kinase that mediates apoptosis signaling with upregulation of interferon-γ, tumor necrosis factor-α, and Fas to initiate apoptosis and anoikis [112,113]. CCN2 assisted DAPK in overcoming the inhibition by MAPK/ERK signaling [52].

p53
As a principal apoptosis regulator of tumors, p53 plays a pivotal role in anoikismediated cell death. p53-dependent anoikis has been demonstrated in several cell lines in lung cancer [44,114]. p53 upregulated cleavage caspase-3 to inhibit integrin α6β4, AKT signaling, and tumor metastasis [115]. XEDAR is an effector downstream of p53. p53 binds to intron 1 of the XEDAR gene and promotes transcription [44]. The expression of p53 and liver kinase B1 (LKB1) is positive feedback; however, loss of p53 or p14 cooperating with mutant Kras results in the inactivation of LKB1 [116][117][118]. Regulation of the p53 pathway is essential for tumor treatment, and some drugs interfere with the p53 pathway, as detailed below.

BIM
The pro-apoptotic protein BIM, a BH3-only protein, is a critical executor of apoptosis in anoikis. BIM disrupts the outer mitochondrial membrane to induce apoptosis, a response to anchorage-independent growth [119]. 14-3-3ζ knockdown in A549 cells was accompanied by upregulation of the pro-apoptotic protein BIM rather than Bad and susceptibility to anoikis. BIM inhibits BCL-2, Bcl-xL, and MCL-1, leading to Bax activation and anoikis [120]. In addition, when RhoA/ROCK senses cell detachment, signals are transmitted to BIM to initiate the apoptosis process [10].

Bit1
BCL-2 inhibitor of transcription 1 (Bit1) is named for the ability to reduce BCL-2 promoter activity. After integrin-or ECM-mediated cell attachment, Bit1 is released from the mitochondria to the cytoplasm and forms a complex with the transcriptional regulator amino-terminal enhancer split (AES) protein to initiate cell death against anchorageindependent growth [121,122]. In addition, the Bit1/AES complex upregulated E-cadherin to inhibit EMT and enhanced anoikis in lung cancer cells in vitro [121,123]. Transducin-like enhancer of split 1 (TLE1) sequestrates AES into the nucleus to reduce the formation of the Bit1/AES complex. Bit1 induced cytoplasmic translocation and degradation of TLE1 during anoikis [122,124,125]. A summary is presented in Figure 5.
amino-terminal enhancer split (AES) protein to initiate cell death against anchorage-independent growth [121,122]. In addition, the Bit1/AES complex upregulated E-cadherin to inhibit EMT and enhanced anoikis in lung cancer cells in vitro [121,123]. Transducin-like enhancer of split 1 (TLE1) sequestrates AES into the nucleus to reduce the formation of the Bit1/AES complex. Bit1 induced cytoplasmic translocation and degradation of TLE1 during anoikis [122,124,125]. A summary is presented in Figure 5.

LKB1
Several studies demonstrated that LKB1 accumulation promotes anoikis in tumors [126]. LKB1 decreases anchorage-independent growth of A549 and HeLa cells, suggesting that LKB1 is a tumor suppressor and anti-metastasis factor [127]. Salt-inducible kinase 1 (SIK1) is an LKB1-dependent kinase that inversely correlates with poor prognosis and distal metastases in breast cancer. The SIK1/LKB1 complex promotes p53-dependent anoikis and suppresses metastasis in lung cancer [114]. Meanwhile, loss of LKB1 is connected to poor prognosis in lung cancer. In LKB1-deficient lung cancer, pleomorphic adenoma gene 1 (PLAG1) mediates the upregulation of a glutaminolysis enzyme, glutamate dehydrogenase 1 (GDH1), and calcium/calmodulin-dependent protein kinase kinase 2 bound

Therapy for Anoikis in Lung Cancer
Investigations in lung cancer models demonstrate the potent anti-tumor activity of treatments such as monoclonal antibodies (mAbs), natural products, chemosynthetic drugs, and multi-component drugs against lung cancer metastasis via activation of anoikis (Table 2 and Figure 6).

Natural Products
Numerous studies suggest that drugs enhancing anoikis are derived from natural products, of which small molecule compounds are the most common, including natural herbs such as Thai medicine, Chinese medicine, and marine natural products. The primary targets of natural products for anoikis include inhibition of MCL-1, AKT, FAK, and EMT.
For MCL-1 and anoikis-related pathways, renieramycin M and ecteinascidin 770, isolated from Thai tunicate Ecteinascidia thurstoni, exhibit promising anticancer activity via a p53-dependent pathway and inhibition of MCL-1 and BCL-2 [148,149]. Imperatorin from Angelica dahurica root was first reported by Pithi et al.; it enhances protein expression of p53 and Bax and reduces MCL-1, which promotes anoikis and cell death sensitization in several lung cancer cell lines [150]. Artonin E is extracted from the bark of Artocarpus gomezianus; it enhances the anoikis of lung cancer cells in a dose-dependent manner by downregulating MCL-1 [151].

Natural Products
Numerous studies suggest that drugs enhancing anoikis are derived from natural products, of which small molecule compounds are the most common, including natural herbs such as Thai medicine, Chinese medicine, and marine natural products. The primary targets of natural products for anoikis include inhibition of MCL-1, AKT, FAK, and EMT.
For MCL-1 and anoikis-related pathways, renieramycin M and ecteinascidin 770, isolated from Thai tunicate Ecteinascidia thurstoni, exhibit promising anticancer activity via a p53-dependent pathway and inhibition of MCL-1 and BCL-2 [148,149]. Imperatorin from Angelica dahurica root was first reported by Pithi et al.; it enhances protein expression of p53 and Bax and reduces MCL-1, which promotes anoikis and cell death sensitization in several lung cancer cell lines [150]. Artonin E is extracted from the bark of Artocarpus gomezianus; it enhances the anoikis of lung cancer cells in a dose-dependent manner by downregulating MCL-1 [151]. FAK is associated with cell adhesion, and inhibition of FAK contributes to anoikis. Sulforaphane is an isothiocyanate found in cruciferous vegetables that promotes anoikis and cell death. Sulforaphane reduces FAK, AKT, and β-catenin and upregulates p21 to induce anoikis in lung cancer cells with wild-type p53 [155]. Pongamol is a chalcone derived from the T. purpurea stem extract that mitigates EMT and permits anoikis through downregulation of the FAK and AKT/mTOR signaling pathways [156]. EPS11 is a purified polysaccharide from a crude extract of marine bacterial polysaccharide; it inhibits the expression of βIII-tubulin at the transcription level, triggering suppression of the downstream effectors phospho-PKB and phospho-AKT in vitro and in vivo [157].
EMT promotes the progression and metastasis of lung cancer, and some natural products target anoikis by inhibiting EMT. Ginsenoside Rg3 promotes anoikis and EMT inhibition via the TGF-β/SMAD pathway in A549 [158]. Ginsenosides Rk1 and Rg5 suppress EMT through downregulation of MMP2/9 activity, inhibitory actions of Smad2/3, and the NF-kB/ERK pathway in A549 cells [159]. Jorunnamycin A is a bis-tetrahydroisoquinoline quinone isolated from the blue sponge Xestospongia sp.; it reduces EMT and anchorageindependent survival by regulating apoptosis-related proteins and EMT markers. p53 and E-cadherin are upregulated after treatment with Jorunnamycin A [160].
Crude extracts of plants and Chinese medicine formulas target anoikis in lung cancer. A polysaccharide extract obtained from rough extraction of persimmon leaves inhibited EMT and anoikis resistance through the canonical TGF-β/SMAD pathway and inhibited the MAPK pathway [171]. Oat avenanthramides inhibited EGFR, suppressing lung cancer cell growth and migration [172]. Jinfukang is a traditional Chinese medicine prescription for lung cancer treatment; it inhibits the integrin/Src pathway, suppressing ECM-receptor interaction and focal adhesion-related genes [173]. Modified Bu-Fei decoction inhibits hypoxia-inducible factor 1α (HIF-1α) signaling and angiogenin-like protein 4 (ANGPTL4) to inhibit A549 cells anoikis and lung metastasis of LLC-bearing mice [174].

Synthetic Products
Chemosynthetic products have more precise targets and lower active concentrations for treating malignant tumors; these include the inhibitors of some targets such as BCL-2 and EGFR. BCL-2 inhibitor ABT-263 acts as adjunctive therapy to promote cell death-related proteins and anoikis [175]. WZ4002, a third-generation EGFR inhibitor, causes anoikis and inhibits lung cancer metastasis [176]. TMPRSS4 serine protease inhibitor KRT1853 promotes anoikis of lung cancer cells by inhibiting the JNK/MAPK, PI3K/AKT, and NF-κB pathways [177]. Nintedanib is available for idiopathic pulmonary fibrosis treatment. Highdose nintedanib (5 uM) promotes anoikis and apoptosis via downregulation of PCNA in NSCLC [178].

Discussion
Anoikis is a critical biological process that antagonizes lung cancer metastasis. Tumor metastasis begins when cells detach from their native environment and adapt to new sites through blood vessels, lymphatics, or body cavities. Anoikis prevents tumor cells from detachment and re-adhesion to new matrices in incorrect locations or the new organism. During this process, the ECM, including collagen IV; laminin 5; fibronectin; and cell membrane proteins including integrins, CEA, EGFR, CDCP1, and CAV1, acts as a sensor and is the first site to receive cell detachment signals. CEA and EGFR engage in crosstalk with integrins and promote metastasis. Integrins can trigger FAK/Src, an essential pathway for inhibiting anoikis. CAV1 is a membrane adapter to kinase Fyn in integrin signaling; it interacts with MCL-1 to inhibit anoikis (Figure 7). p66 shc is a focal adhesion regulatory protein that initiates apoptosis signals for anoikis. Apoptosis-related proteins (i.e., BIM, P53, DAPK, and caspases) are critical targets driving anoikis. Inhibitors of apoptosis proteins MCL-1, Bit, and BCL-2 suppress anoikis. CEA, TrkB, FAK/Src, and βIII-tubulin inhibit anoikis through upregulation of the PI3K/AKT pathway. Next, growth factors negatively regulate anoikis, and TGF-β activates integrins to trigger intracellular apoptosis resistance. EFGR and TrkB activate ERK/MAPK or Ras/Raf/ERK signaling pathways to counteract anoikis. Furthermore, cytoskeleton regulator RhoA and the downstream effector ROCK antagonize Ras and FAK signaling and promote apoptotic signaling (Figure 8).
Because of evidence of anoikis reducing metastasis, preclinical investigations in lung cancer focus on targeting anoikis using novel small molecule compounds (natural and synthetic products), mAbs, and repurposed FDA-approved compounds. Therapeutic targets include increased p53 and inhibition of TGF-β/SMAD, FAK, AKT, and MCL-1. Despite several preclinical studies on anoikis-related inhibitors, a gap urgently needs to be filled, including an analysis of compounds that combat lung cancer metastases in vivo.

Discussion
Anoikis is a critical biological process that antagonizes lung cancer metastasis. Tumor metastasis begins when cells detach from their native environment and adapt to new sites through blood vessels, lymphatics, or body cavities. Anoikis prevents tumor cells from detachment and re-adhesion to new matrices in incorrect locations or the new organism. During this process, the ECM, including collagen IV; laminin 5; fibronectin; and cell membrane proteins including integrins, CEA, EGFR, CDCP1, and CAV1, acts as a sensor and is the first site to receive cell detachment signals. CEA and EGFR engage in crosstalk with integrins and promote metastasis. Integrins can trigger FAK/Src, an essential pathway for inhibiting anoikis. CAV1 is a membrane adapter to kinase Fyn in integrin signaling; it interacts with MCL-1 to inhibit anoikis (Figure 7). p66 shc is a focal adhesion regulatory protein that initiates apoptosis signals for anoikis. Apoptosis-related proteins (i.e., BIM, P53, DAPK, and caspases) are critical targets driving anoikis. Inhibitors of apoptosis proteins MCL-1, Bit, and BCL-2 suppress anoikis. CEA, TrkB, FAK/Src, and βIII-tubulin inhibit anoikis through upregulation of the PI3K/AKT pathway. Next, growth factors negatively regulate anoikis, and TGF-β activates integrins to trigger intracellular apoptosis resistance. EFGR and TrkB activate ERK/MAPK or Ras/Raf/ERK signaling pathways to counteract anoikis. Furthermore, cytoskeleton regulator RhoA and the downstream effector ROCK antagonize Ras and FAK signaling and promote apoptotic signaling (Figure 8). Because of evidence of anoikis reducing metastasis, preclinical investigations in lung cancer focus on targeting anoikis using novel small molecule compounds (natural and synthetic products), mAbs, and repurposed FDA-approved compounds. Therapeutic

Conclusions
Anoikis plays an important role in lung cancer metastasis and is associated with tumor progression and therapy failure. The composition of the extracellular matrix, cell adhesion-related membrane proteins, cytoskeletal regulators, and epithelial-mesenchymal transition are involved in the process of anoikis, and the initiation of apoptosis signals is a critical step in anoikis. Several natural and synthetic products, including artonin E, imperatorin, oroxylin A, lupalbigenin, sulforaphane, renieramycin M, avicequinone B, and carbenoxolone, exhibit pro-anoikis potential. This review provides an overview of the major regulators and mechanisms of anoikis in lung cancer and discusses the therapeutic potential of targeting anoikis in the treatment of lung cancer.