The N terminus of PHLPP1 has about 50 kDa of unknown function structure called N-terminal extension (NTE). Either directly binding PHLPP1 or indirectly binding through other scaffold proteins can pass through this domain (24). The function of NTE is to switch the PHLPP1 protein interaction network during mitosis (25). NTE is a substrate for cyclin-dependent kinases 1 (Cdk1) and dissociates from plasma membrane scaffolds such as Scribble (26). When NTE approaches and acquires centromeric and mitotic spindle proteins, it affects cell mitosis and inhibits normal cell division cycles, which is associated with cancer progression and poor prognosis (27–29). Despite being evolutionarily low conserved, NTE is necessary for the recently discovered dephosphorylation of PHLPP1 transcriptional regulatory substrate signal transducer and activator of transcription 1 (STAT1) in colitis (24) ( Figure 1 ).

PH domain is present in many kinases, phospholipase C, nucleotide exchange factors, GTPases and the GTP-binding proteins which interact with many proteins (30). The PH domain of PHLPPs, a globular structure containing about 120 amino acids, is acquired relatively late in evolution compared to other domains and is found only in mammals (31, 32) ( Figure 1 and section 3.2 for the role of this domain in LC).

The LRR domain of PHLPPs isozymes is characterized by multiple repeated leucine residues at the same location. It consists of 18 repeated amino acid sequences (33). This domain regulates transcription of receptor tyrosine kinase (RTK) such as Epidermal growth factor receptor (EGFR) (34) ( Figures 1 , 2 ), and interact with KRAS to block the activation of ERK (35) ( Figure 1 and section 3.3).

The main role of the PP2C domain is to resist the action of common serine/threonine phosphatase inhibitors, resulting in drug resistance (36). Both PHLPP1 and PHLPP2 are members of the PP2C family and are insensitive to most common phosphatase inhibitors (37). Thus, PHLPPs can act as a tumor suppressor proteins as a member of the PP2C family, which is mainly involved in cell growth and stress signaling pathways (38). In PHLPPs, the catalytic effect of PP2C domain alone is low (22), but when it co-exists with the PDZ-binding motif, it become more essential for scaffold binding and AKT dephosphorylation (37) ( Figure 1 ).

In PHLPPs family members, the PDZ ligand is critical for the dephosphorylation of AKT in cells (5). PDZ ligands of PHLPP1 and PHLPP2 bind to scaffold Na+/H+ exchanger Regulatory Factor 1 (NHERF1) (7) due to localization of phosphatases near the membrane, where they could dephosphorylate AKT (39). Both PHLPP1 and PHLPP2 selectively dephosphorylate the same site on AKT despite acting on different AKT subtypes (37, 40). See section 3.1 ( Figure 1 ).

PI3K contributes to a variety of cellular processes including cell growth, migration, differentiation, and proliferation by recruiting proteins with PH domains to the plasma membrane (41) ( Figure 2 ). One of the most critical and versatile protein kinases in higher eukaryotes is protein kinase AKT (42). Many of AKT’s downstream related substrates have been shown to be involved in cell metabolism, cell survival, cell proliferation and migration (43). Stimulating activation of the PI3K/AKT/mTOR pathway is a key event in the development of cancer especially lung cancer (44–46). Thus, it causes cancer insensitive to treatment ( Figure 3 ).

Based on the past research, PHLPPs mainly produce dephosphorylated AKT through the PDZ domain (5). The AKT isomer was investigated and found that PHLPP1 selectively dephosphorylated AKT2, whereas PHLPP2 selectively dephosphorylated AKT1 (47). The ultimate dephosphorylation outcome can inhibit tumor cell growth, thereby prolonging patient survival (48) (The specific role of PHLPPs in LC through this pathway was described in section 4).

PKC is widely involved in cell survival, mitosis, motility, and invasion. This kinase was initially described as an oncogene and subsequently identified as a cancer biomarker, showing overexpression of PKC in multiple epithelial tumors, including lung cancer (49). Abnormal expression of PKC contributes to the initiation and progression of cancer (50, 51). By database analysis, PKC expression was associated with poor prognosis in KRAS-mutated LUAD (52). PKC is also a highly specific oncogene, it is a key factor in the sensitivity of the body to chemical carcinogens and the induction of carcinogenesis by gene mutation. In the absence of PKC, the number of KRAS-driven and chemical carcinogen-induced lung diseases was reduced(52). More, PKC is essential for the normal physiological response of many cells in lung including permeability, migration, proliferation, apoptosis, and secretion (53, 54).

As showed the PKC function above, we consulted the relationship between PKC and PHLPP. Both PHLPP1 and PHLPP2 can dephosphorylate the hydrophobic motif of PKC, leading unstable and degradation of PKC protein (31, 55). Loss of PHLPPs phosphatase function could lead to the increasing of PKC protein level, thus promoting tumorigenesis (31, 56). Depletion of PHLPPs in cells leaded to the AKT phosphorylation and increased total PKC levels (55, 57). A study showed that PKCα was fully phosphorylated at the PHLPP site, and PHLPP1 levels were negatively correlated with PKC in more than 5,000 tumor patients (58). Other studies also showed that PHLPP affected disease progression through the PKC pathway (59). Those findings provided evidence that PHLPP was a potential tumor suppressor ( Figures 2 , 3 ).

During disease progression, upstream RTK signals such as EGF stimulate tyrosine kinase receptors, which in turn cause activation of RAS, and further activates RAF/MEK/ERK proteins through kinase cascades (60). The MAP kinase (MAPK)/ERK pathway has been shown to play an important role in the growth and differentiation of tumor cells and to play an effective carcinogenic role in LUAD (61–63). In colorectal cancer, one study reported that both PHLPP1 and PhLPP2 dephosphorylated RAF1, thereby inhibiting colorectal cancer proliferation. When PHLPP1 and 2 were knocked out, the expression of epithelial-mesenchymal transition (EMT) markers was increased, which promoted the migration and invasion of colorectal cancer, tumor proliferation and progression and meanwhile negatively regulate MEK signaling pathways (64).

Simultaneous mutations of v-Raf murine sarcoma viral oncogene homolog B (BRAF) and KRAS, two key oncogenes in RAS/RAF/MEK/ERK signaling pathway, had been found in NSCLC (65, 66). Previous studies have reported that PHLPP1 could directly interact with KRAS through its LRR domain to negatively regulate ERK pathway (35, 67). PHLPPs also reduces ERK phosphorylation. Our previous patient studies have shown that highly expressed PHLPP1 can have a longer duration of EGFR- TKI resistance, which mainly through MAPK/ERK signaling pathway. It was found that PHLPP1 had a negative correlation with phosphorylated ERK expression in LUAD patients. Similarly, in vitro studies, it was found that overexpression of PHLPP1 inhibited EGFR-TKI resistance in non-small cell lung cancer via the MAPK/ERK pathway (20, 21). See section 4 ( Figure 3 ).

However, the current research is still limited. Since the discovery of PHLPPs, a lot of studies have been conducted on illuminating of its substrates, including STAT1, MST1, S6K1 and so on (24, 68, 69). Although these substrates were associated with the development of lung cancer (70–72), there was no direct evidence that they were associated with PHLPPs in LC. In addition, in a global PHLPP knocked out (PHLPP^-/-) mouse model, researcher has focused more on the gastrointestinal tract, including the progress of cancer and the changes of immune cells in the immune microenvironment.

Since its discovery, PHLPPs has been proved to play a role in a variety of solid tumors, including colorectal (6, 64, 80), prostate (7, 81, 82), breast (83), and so on. Among lung cancers, NSCLC accounts for a larger proportion, with a higher incidence of LUAD, accompanied by the most common EGFR mutation. Based on the above basic research effect of PHLPPs, we summarize its clinical application in LUAD.

Radiotherapy is the most effective cytotoxic therapy for local solid carcinoma (116–118). Radiation therapy is the primary treatment for patients with inoperable NSCLC and has a better therapeutic prospect than surgery (119). Based on these good clinical phenomena, more and more people have conducted new research on the mechanism under radiotherapy, trying to find more accurate tumor treatment strategies. Radiation therapy can inhibit tumor growth infiltration by affecting the growth cycle, proliferation of tumor cells and tumor angiogenesis, DNA damage or immune activation and other related pathways (120–125). However, radiation resistance during treatment limits the effectiveness of radiation therapy in patients, one of the possible causes of radio-resistance is the abnormal activation pathways in tumor cells during radiotherapy, such as the RTKs-related protein PI3K/AKT (126) which was shown that abnormal AKT activation can promote DNA damage repair and enhance cell resistance (127).

It is well known that the direct target of PHLPPs is AKT, which inhibits tumor proliferation and invasion through dephosphorylation of AKT. Previous study demonstrated that radiation could affect USP46 expression levels and thus altered the expression of PHLPP1 and AKT (19). However, the role of PHLPPs in radiation therapy has not yet been explored. Targeted inhibition of phosphatase activity may be another option to enhance tumor radiosensitivity (128). We speculate that PHLPPs is involved in radiation-induced DNA damage and thus affects tumor proliferation. And that’s the goal we’re going to explore.

In addition, radiation therapy also caused changes in the PKC pathway. He et al. had demonstrated that ionizing radiation can induce classical PKC (cPKC) activation, thereby promoting RAS/ERK activation (129) and finally apoptosis was promoted and apoptosis-like related markers were changed (130). We have previously discussed the relationship between PHLPP and PKC (see section 3.2). On this basis, the hypothesis that whether radiotherapy can directly act on PHLPPs and induce apoptosis has proposed a new direction for us to explore the effect of PHLPPs in lung cancer ( Figure 4 ).

In addition, immunotherapy has also been a great success. There have been great advances in treating cancer through immunotherapy over the past decade. The effective way to achieve anti-tumor immunity is to block the bridge between cancer cells and immune cells to activate the body’s immune response. Existing immune checkpoint inhibitors can restore the killing effect of immune cells on tumor cells, especially in lung cancer has entered clinical use (131). However, there are different immune cells in the tumor microenvironment. How to reverse the immunosuppressive cells in the tumor microenvironment to the activated state to kill tumor cells has also become the direction of future research.

Macrophages, as the primary response cells to innate immune response, can use pattern recognition receptors (PRRs) to accurately recognize foreign pathogens and initiate inflammatory responses (132). Therefore, macrophages play an important role in maintaining tissue homeostasis after tissue injury. In a previous study, mice with whole-body knockouts of PHLPP1 showed strong resistance to the lipopolysaccharide (LPS), a component of Gram-negative bacteria, and significantly increased survival time compared with the control group mice. The main reason was that macrophages with PHLPP1 knockout were able to dephosphorylate signal transducers and activators of transcription 1 (STAT1) after being stimulated by LPS, thus increasing the inflammatory response(24). In addition, another mechanism by which LPS enhanced macrophage inflammatory response has been reported due to the regulation of PHLPP1 expression by specificity protein 1 (SP1)(133).

In colitis, the absence of PHLPPs protects the mice from inflammation (16). An in-depth study showed that the mucosal immunity was improved, the migration function of mucosal neutrophils was enhanced in a systemic PHLPPs knockout mouse model. In the same model, PHLPPs was helpful to maintain neutrophilic homeostasis and alleviate colitis reaction (134). In CD4⁺T cells, inhibition of PHLPP1/2 expression can reduce the induction of Treg cells (18).

In NSCLC, a variety of immune cells in the tumor microenvironment participate in the treatment of immune checkpoint inhibitors (ICIs)(135). Although, as previously described, PHLPPs can act as an immune activator or suppressor in immune cells (23), the role of PHLPPs in immunotherapy in lung cancer remains unanswered. This is also a potential mechanism for the future treatment of lung cancer by PHLPPs as a new target ( Figure 4 ).

There are many causes of lung cancer, including genetic factors, abnormal gene activation, harmful chemicals in the environment, smoking and so on(136), among which smoking is the main cause of lung cancer. And about 85 percent of lung cancer cases are caused by smoking (137). There was already evidence that particles released after smoking activate nuclear factor-kappaB (NF-κB) via myristoylated alanine-rich C kinase substrate (MARCKS) protein, which promotes tumorigenesis and proliferation and survival of lung cancer cells (138, 139). NF-κB was an important regulator of inflammatory processes and also plays a role in lung cancer(140). A previous study showed that deletion of PHLPP2 led to an increase in p-NF-κB p65 in prostate cancer with a PTEN mutation(81). This provided support for our hypothesis that PHLPPs might influence the progression of lung cancer through the NF-κB inflammatory signaling pathway ( Figure 4 ).

Cell death forms include apoptosis, necrosis, pyroptosis, autophagy and so on (141–144). As we know, one of the reasons why tumors proliferation and progress is because they inhibit normal cell death. So how to induce different cell death patterns is also an idea of tumor therapy. The newly discovered iron-related cell death named ferroptosis is a new way of cell death (145), which related to the accumulation of reactive oxygen species(ROS) in cytoplasm, characterized by iron accumulation and lipid peroxidation. Ferroptotic cell death morphologically presents a smaller mitochondria but with increased mitochondrial membrane density and reduced or non-existent mitochondria crista, and outer mitochondrial membrane rupture than normal cell (146). In lung diseases including acute lung injury, chronic obstructive pulmonary disease, pulmonary fibrosis, radiation-induced lung injury, asthma, ferroptosis has shown to play a crucial role, and that provide a new treatment ideas(147). Our recent study showed that PHLPP2 regulated ferroptosis through nuclear factor erythroid 2-related factor 2 (Nrf2) pathway to affect apoptosis in LUSC. In addition, PHLPP2 can work with RSL3, a glutathione peroxidase 4 (GPX4) inhibitor, to enhance the inhibition of GPX4 activity, thereby increasing intracellular ROS accumulation and inducing Ferroptosis. However, the interaction between PHLPP2 and ferroptosis has not been explored, including nuclear receptor coactivator 4 (NCOA4), a key protein in iron metabolism pathway, and GPX4/acyl-CoA synthetase long-chain family member 4 (ACSL4), key proteins in Glutathione (GSH) metabolism pathway, and solute carrier family 7 (SLC7A11), a key protein in ROS metabolism pathway(148). The specific role of PHLPPs in inducing ferroptosis in lung cancer cells may provide a new direction for radiotherapy resistance patients in the future, which may be a new site of action for future research ( Figure 4 ).