Implications of Phosphatase and Tensin Homolog in NSCLC: Comparison
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Please note this is a comparison between Version 2 by Jason Zhu and Version 1 by Ravi Sahu.

Lung cancer remains one of the major human malignancies affecting both men and women worldwide, with non-small cell lung cancer (NSCLC) being the most prevalent type. Multiple mechanisms have been identified that favor tumor growth as well as impede the efficacy of therapeutic regimens in lung cancer patients. Among tumor suppressor genes that play critical roles in regulating cancer growth, the phosphatase and tensin homolog (PTEN) constitutes one of the important family members implicated in controlling various functional activities of tumor cells, including cell proliferation, apoptosis, angiogenesis, and metastasis.

  • non-small cell lung cancer
  • PTEN
  • cancer chemotherapy

1. In Vitro and In Vivo Studies

Several studies have used in vitro culture systems and mouse models to define the functional significance of PTEN in NSCLC. In one study, Lu and colleagues investigated the crosstalk of PTEN with hTERT and the PI3K/AKT pathway, using the lung adenocarcinoma A549 cell line overexpressing wild-type or mutant PTEN, or the PTEN siRNA approach [75][1]. The result showed that PTEN plays a vital role in suppressing cell proliferation via inducing cell cycle arrest and apoptosis. In comparison to the control group, it was found that the mRNA and protein levels of hTERT were lower in the A549 cell line transfected with wild-type PTEN. Also, in A549 cells transfected with PTEN-siRNA and hTERT mRNA, protein levels were significantly higher than in the control group. These findings show that PTEN inhibits hTERT expression in NSCLC cells. The PTEN mRNA and protein levels increased when the A549 cells were transfected with the phosphatase-dead PTEN mutant, but there was no change in hTERT expression levels, indicating that the phosphatase-dead PTEN mutant is nonfunctional and that only wild-type PTEN suppresses hTERT expression. Mechanistically, it has been observed that PTEN suppressed cell proliferation by inhibiting the PI3K/AKT/hTERT pathway. Overall, these findings indicated that the downregulation of the PI3K/AKT/hTERT pathway is one of the PTEN mechanisms that acts as a tumor suppressor in lung cancer [75][1].
Moreover, the PTEN expression in lung cancer cells is regulated by deubiquitylase Ataxin-3 [76][2]. For the identification of deubiquitylates (DUBs), Sacco and colleagues used an unbiased siRNA screening approach and found that PTEN expression is affected by the PI3K pathway. The members of the DUBs family consist mainly of ATXN3, ATXN3L, and JOSD1, as well as other factors, like PTENP1 and PTEN transcripts, and its RNA levels increased markedly as the reduction in each of the DUBs occurred [76][2]. The degradation of the PTEN protein did not affect the DUBs. Importantly, the PTEN induction observed in response to the ATXN3 siRNA was found to sufficiently downregulate AKT phosphorylation and, hence, the PI3K signaling. Also, histone deacetylase inhibitors (HDACi) have been suggested as potential mediators of PTEN transcriptional reactivation in NSCLC. The authors found that although PTEN exhibited a very limited response to the broad-spectrum HDACi, vorinostat (SAHA), in A549 cells, its combination with ATXN3 depletion enhanced PTEN induction in an additive manner. Similarly, these interventions additively decreased the cell viability. These findings indicated that ATXN3 acts as an autonomous target for therapeutic intervention for lung cancer, which is associated with an epigenetic downregulation of PTEN [76][2].
In addition, pemetrexed treatment for lung cancer cells has also been documented to inhibit the PI3K pathway. In a study, Li and colleagues demonstrated that PTEN overexpression can increase the efficacy of pemetrexed in the A549 NSCLC cell line [77][3]. Importantly, the combination of pemetrexed with PTEN overexpression inhibited the AKT signaling pathway; however, the mTOR signaling pathway was found to be activated, which resulted in the induction of apoptosis-associated genes, including p53, Bcl2, and BAX. In addition, the results suggested that treatment with pemetrexed combined with PTEN overexpression can inhibit the aerobic oxidation of glucose, which decreases the energy supply of cancer cells, leading to increased apoptosis. Overall, the data indicated that pemetrexed treatment inhibits the proliferation of NSCLC cells via targeting the PI3K/AKT/mTOR signaling pathway and carbohydrate metabolism, and thus, it represents a novel therapeutic strategy for the treatment of NSCLC [77][3].
Importantly, Noro and colleagues determined how PTEN inactivation can affect tumor progression and drug resistance in lung cancer [78][4]. By using a panel of lung cancer cell lines, the authors examined the levels of PTEN expression at both the mRNA and protein levels and their genetic and epigenetic status. Low expression of the PTEN protein was documented in six cell lines out of the twenty-five cell lines tested. Out of the six cell lines having low PTEN expression, the genomic analysis of the QG56 and N23 cell lines showed homozygous deletions of the PTEN gene. By using a methylation-specific PCR, the authors showed that the PC10 and PC14 cell lines have hyper-methylation of the PTEN gene promoter. After treatment with the demethylating agent, 5-aza-2′deoxycytidine (5-AZA), and the histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), the authors observed that the gefitinib and TSA combination induced significant growth inhibition in two gefitinib-resistant cell lines. Overall, these findings suggested that the combination of gefitinib with the demethylating agent, 5-AZA, and TSA could be beneficial for treating NSCLC [78][4].
As PTEN deletion has been shown to occur within the tumor cells residing in the airway basal cells, Malkoski and colleagues performed a study using a mouse model to examine if the loss of PTEN in airway basal cells can initiate tumor formation or increase squamous cell carcinoma formation [80][5]. The authors have previously established that targeting KrasG12D activation and transforming growth factor β receptor type II (TGFβRII) deletion to airway basal cells via a keratin promoter resulted in the development of both adenocarcinoma and squamous cell carcinoma in the lungs. The authors found that when PTEN deletion occurs in targeting basal cells, it can initiate both lung adenocarcinoma and squamous cell carcinoma formation. Although PTEN deletion is a weaker tumor initiator than KrasG12D, with low tumor multiplicity and long latency, tumors initiated by PTEN deletion were larger and more malignant than KrasG12D-initiated tumors. When genetic modifications were focused on a particular cell, the fact that PTEN loss did not enhance lung SCC development in comparison to KrasG12D activation implies that the initiating genetic event does not determine tumor histology. These findings also indicated that conducting airway basal cells can give rise to a variety of NSCLC tumors [80][5].
Along similar lines, Yu and colleagues determined the mechanisms and functional significance of the abnormal expression of PTEN using various in vitro and in vivo models of NSCLC [79][6]. The authors demonstrated that PTEN knockdown induced increased proliferation of the H1975, A549, HCC827, and H1650 cell lines, including a colony formation assay, and resulted in the enhanced growth of subcutaneously implanted tumor xenografts. The overexpression of PTEN rescued the loss of the PTEN phenotypes, while suppression of the PTEN expression via the shRNA approach was found to increase the number of metastatic tumors. These studies also revealed that the integrin αVβ6 signaling pathway was affected by PTEN [79][6].
Notably, protein ubiquitination plays a dynamic role in modulating protein stability, including in PTEN, and it requires ubiquitin-specific proteases (USPs) or deubiquitinases (Dubs) to hydrolyze the ubiquitin molecules from their substrates. In a study, He and colleagues determined the effect and mechanism of USP10 as a tumor suppressor Dub of PTEN [81][7]. The authors previously established that an E3 ubiquitin ligase, tripartite motif-containing 25 (TRIM25), induces the K63-linked polyubiquitination of PTEN and inhibits its phosphatase activity [85][8], which provided the rationale to explore Dubs in this effect. Using immunoprecipitation/immunoblotting assays, the authors found USP10 to be the most potent Dub in suppressing PTEN K63-linked polyubiquitination mediated by TRIM25. Further studies confirmed that USP10 interrupted the interaction between PTEN and TRIM25 yet did not affect the mRNA or protein stability of PTEN [81][7]. Importantly, USP10 expression was downregulated/low in the NSCLC cell lines and in the primary NSCLC tissues compared to the normal human bronchial epithelial cells and matched normal tissues. Mechanistic studies demonstrated that USP10 suppresses the activation of the AKT/mTOR pathway via decreasing the K63-linked polyubiquitination of PTEN, which resulted in the inhibition of the cell viability, proliferation, and migration of the A549 and H1299 NSCLC cell lines. Overall, these studies demonstrated that USP10 targets the PTEN/AKT/mTOR signaling pathway in NSCLC by preventing K63-linked polyubiquitination of PTEN and, thus, acts as a tumor suppressor.
Of importance, the role and mechanisms of the RNA-binding motif protein 10 (RBM10), an alternative splicing regulator that plays an important role in regulating proliferation and apoptosis, was determined in NSCLC [82][9]. An analysis of NSCLC tissues from patients and the NSCLC A549 and H1299 cell lines revealed lower expression of RBM10 as compared to paired paracancerous tissues and the human bronchial epithelial cell line (BEAS-2B). The overexpression of RBM10 was suppressed, and its silencing enhanced invasion and migration, as well as EMT-related protein expression, indicating its tumor suppressor activity. Mechanistically, these effects were found to be mediated via RBM10-induced PTEN expression and inhibition of the activation of the PI3K/AKT/mTOR pathway [82][9]. Furthermore, clip-seq analysis demonstrated that RBM10 interacts with 353 long non-coding RNAs (lncRNAs), and among these, it binds to the nuclear enriched abundant transcript 1 (Neat1) with higher affinity and also regulates its splicing variants. Importantly, RBM10 overexpression was reduced, and its silencing increased Neat1 expression, indicating that RBM10 regulates the alternative splicing (AS) of Neat1 [82][9]. Further studies confirmed that induced RBM10 increased PTEN expression and decreased PI3K/AKT/mTOR activation and was mediated via Neat1 splicing variants. These studies indicated that RBM1 targets the PTEN/PI3K/AKT/mTOR/Neat1 axis to regulate the growth of NSCLC cells.
Along similar lines, studies by Cai and colleagues demonstrated that casein kinase 1 alpha 1 (CK1α), a negative regulator of the Wnt pathway, induces PTEN stabilization and activity by preventing PTEN and NEDD4-1 binding and inhibiting PTEN polyubiquitination and phosphorylation [83][10]. This effect resulted in the inhibition of AKT activity and the upregulation of the FOXO3a-induced transcription of the E1 enzyme, Atg7, which plays a crucial role in inducing autophagy. Mechanistic studies demonstrated that the CK1α/PTEN/FOXO3a/Atg7 axis inhibits the growth of A549 NSCLC cells and A549 lung tumor xenografts via inducing autophagy and that blocking CK1α-induced autophagy mediates oncogenic HRasV12. Importantly, high expression of CK1α, PTEN, and Atg7 in human NSCLC tissues was found to be associated with increased overall survival and vice versa. Overall, these findings indicated that PTEN interacts with CK1α, which maintains PTEN stability and activity to induce autophagy in NSCLC.
In one study, He and colleagues determined the role and mechanism of protein tyrosine phosphatase interacting protein 51 (PTPIP51) as a tumor suppressor and its interaction with PTEN in NSCLC [84][11]. Using an integrated bioinformatics approach and NSCLC tissue samples and their matched normal tissue samples, the authors demonstrated that PTPIP51 expression is downregulated in NSCLC, and its low expression was associated with poor patient survival [84][11]. An analysis of PTPIP51 in NSCLC patients harboring EGFR mutations and treated with a targeted kinase inhibitor showed its significantly increased expression, which was associated with an overall improved objective response rate (ORR). To determine the functional significance of PTPIP51 in NSCLC, the authors overexpressed PTPIP51 in the PC9 and A549 cell lines and found that it impairs in vitro cell proliferation, induces apoptosis, and enhances the sensitivity of gefitinib. The in vivo studies with a patient-derived xenograft (PCX) mouse model demonstrated that the intratumoral injection of Ad-PTPIP51 inhibits tumor growth and also increases the sensitivity of gefitinib. Mechanistic studies demonstrated that PTPIP51 not only inhibits EGFR activation but the total EGFR protein levels via promoting its ubiquitylation and subsequent degradation. As PTEN plays a crucial role in EGFR degradation, further studies showed that PTPIP51 directly interacts with PTEN and induces its activation via the CK2 protein kinase and that PTPIP51-PTEN-CK2 complex promotes EGFR degradation [84][11]. Overall, these findings demonstrated that PTPIP51 acts as a tumor suppressor in NSCLC, and its effects were mediated via PTEN activation and EGFR degradation.

2. Clinical Studies

Several studies have investigated the impacts of somatic mutations in PTEN in NSCLC patients. In one study, Jin and colleagues determined the correlation between PTEN mutations and EGFR, KRAS, and TP53 mutations in tumor samples collected from a large cohort of NSCLC patients with or without smoking status [86][12]. The genetic analysis of the various exons of these genes showed that the prevalence of PTEN mutations was 4.5% (eight out of one hundred and seventy-six cases) of patients having a history of smoking. Importantly, 50% of the NSCLC patients with PTEN mutations also had P53 gene mutations. However, EGFR mutations were found to be infrequent (one out of eight cases) among the NSCLC patients harboring PTEN mutations, and none of these patients had KRAS gene mutations. These findings indicated that a subset of PTEN mutation-positive patients exhibit a positive correlation with the P53 and EGFR genes [86][12].
In addition, Sos and colleagues used a vast collection of genomically defined NSCLC cell lines to identify genomic traits that distinguish EGFR-dependent from EGFR-independent EGFR-mutant lung tumor cells. The authors used a combination of computational, biochemical, and cellular methods to discover new mechanisms that uncouple EGFR-dependent tumors from their downstream signaling. The computational genomics analyses found PTEN homozygous deletion to be a candidate for EGFR inhibitor resistance. The functional studies revealed that PTEN loss causes a considerable decrease in apoptosis sensitivity in EGFR-mutant cells by activating AKT and EGFR, and it was hypothesized that ERK activation in PTEN-deficient cells leads to the increased transcription of EGFR ligands, such as amphiregulin [87][13].
In another study, Yoo and colleagues evaluated whether surgically resected primary NSCLC and the loss of PTEN expression correlates with clinicopathological parameters or is related to EGFR gene status in NSCLC [88][14]. The authors analyzed 288 samples for PTEN expression and its correlation with the clinicopathological parameters. It was found that the loss of PTEN expression was associated with 42.4% of the samples, and of those, 28.6% were adenocarcinoma, 66.7% were squamous cell carcinoma, and 38.1% were of an NSCLC subtype. The loss of PTEN expression was significantly associated with smoking, male gender, larger tumor size, and a high pathological stag, while the loss of PTEN expression, age, performance status, and pleural invasion showed no correlation. The survival analysis showed that the progression-free survival was shorter in the group that had a loss of PTEN expression compared to the PTEN intact group. In addition, it was found that PTEN expression was not associated with EGFR status, such as the EGFR copy number or mutation [88][14].
Of importance, Cumberbatch and colleagues found that clinical SCC specimens had a significantly increased expression of PI3Kβ and reduced/loss of PTEN expression compared to adenocarcinoma specimens [89][15]. Detailed correlative analyses of individual patient samples revealed a significantly greater proportion of SCC with higher PI3Kβ and lower PTEN expression. Overall, the authors identified, for the first time, a subset of NSCLC, with the more prevalent SCC having an elevated expression of PI3Kβ, which was accompanied by a reduction or loss of PTEN, for whom selective PI3Kβ inhibitors may be predicted to achieve greater clinical benefit [89][15].
Along similar lines, Panagiotou and colleagues conducted a study to quantitatively analyze the PTEN protein expression in NSCLC. A sample of 61 NSCLC cases was used in the study; about 80% of the cases were males, while 20% of the cases were females. A total of 57% of patients had adenocarcinoma, and 42% had squamous cell carcinoma. Digital image analysis was conducted to analyze the PTEN protein quantitively, and it was found that patients who developed a progressive loss of PTEN expression appeared to have a higher rate of metastases than those with PTEN overexpression. Therefore, in NSCLC, PTEN deregulation was associated with an aggressive phenotype, and this is an important factor in determining a tailored targeted therapy regimen, especially when combined with HER2/PI3K inhibitors [90][16].

References

  1. Lu, X.-X.; Cao, L.-Y.; Chen, X.; Xiao, J.; Zou, Y.; Chen, Q. PTEN Inhibits Cell Proliferation, Promotes Cell Apoptosis, and Induces Cell Cycle Arrest via Downregulating the PI3K/AKT/HTERT Pathway in Lung Adenocarcinoma A549 Cells. BioMed Res. Int. 2016, 2016, 2476842.
  2. Sacco, J.J.; Yau, T.Y.; Darling, S.; Patel, V.; Liu, H.; Urbé, S.; Clague, M.J.; Coulson, J.M. The Deubiquitylase Ataxin-3 Restricts PTEN Transcription in Lung Cancer Cells. Oncogene 2014, 33, 4265–4272.
  3. Li, B.; Zhang, J.; Su, Y.; Hou, Y.; Wang, Z.; Zhao, L.; Sun, S.; Fu, H. Overexpression of PTEN May Increase the Effect of Pemetrexed on A549 Cells via Inhibition of the PI3K/AKT/MTOR Pathway and Carbohydrate Metabolism. Mol. Med. Rep. 2019, 20, 3793–3801.
  4. Noro, R.; Gemma, A.; Miyanaga, A.; Kosaihira, S.; Minegishi, Y.; Nara, M.; Kokubo, Y.; Seike, M.; Kataoka, K.; Matsuda, K.; et al. PTEN Inactivation in Lung Cancer Cells and the Effect of Its Recovery on Treatment with Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors. Int. J. Oncol. 2007, 31, 1157–1163.
  5. Malkoski, S.P.; Cleaver, T.G.; Thompson, J.J.; Sutton, W.P.; Haeger, S.M.; Rodriguez, K.J.; Lu, S.-L.; Merrick, D.; Wang, X.-J. Role of PTEN in Basal Cell Derived Lung Carcinogenesis. Mol. Carcinog. 2014, 53, 841–846.
  6. Yu, Y.X.; Wang, Y.; Liu, H. Overexpression of PTEN Suppresses Non-Small-Cell Lung Carcinoma Metastasis through Inhibition of Integrin AVβ6 Signaling. Am. J. Transl. Res. 2017, 9, 3304–3314.
  7. He, Y.; Jiang, S.; Mao, C.; Zheng, H.; Cao, B.; Zhang, Z.; Zhao, J.; Zeng, Y.; Mao, X. The Deubiquitinase USP10 Restores PTEN Activity and Inhibits Non-Small Cell Lung Cancer Cell Proliferation. J. Biol. Chem. 2021, 297, 101088.
  8. He, Y.-M.; Zhou, X.-M.; Jiang, S.-Y.; Zhang, Z.-B.; Cao, B.-Y.; Liu, J.-B.; Zeng, Y.-Y.; Zhao, J.; Mao, X.-L. TRIM25 Activates AKT/MTOR by Inhibiting PTEN via K63-Linked Polyubiquitination in Non-Small Cell Lung Cancer. Acta Pharmacol. Sin. 2022, 43, 681–691.
  9. Cong, S.; Di, X.; Li, R.; Cao, Y.; Jin, X.; Tian, C.; Zhao, M.; Wang, K. RBM10 Regulates Alternative Splicing of LncRNA Neat1 to Inhibit the Invasion and Metastasis of NSCLC. Cancer Cell Int. 2022, 22, 338.
  10. Cai, J.; Li, R.; Xu, X.; Zhang, L.; Lian, R.; Fang, L.; Huang, Y.; Feng, X.; Liu, X.; Li, X.; et al. CK1α Suppresses Lung Tumour Growth by Stabilizing PTEN and Inducing Autophagy. Nat. Cell Biol. 2018, 20, 465–478.
  11. He, M.; Wang, X.; Chen, W.; Zhang, J.; Xiong, Y.; Cao, L.; Zhang, L.; Zhao, N.; Yang, Y.; Wang, L. PTPIP51 Inhibits Non-Small-Cell Lung Cancer by Promoting PTEN-Mediated EGFR Degradation. Life Sci. 2022, 297, 120293.
  12. Jin, G.; Kim, M.J.; Jeon, H.-S.; Choi, J.E.; Kim, D.S.; Lee, E.B.; Cha, S.I.; Yoon, G.S.; Kim, C.H.; Jung, T.H.; et al. PTEN Mutations and Relationship to EGFR, ERBB2, KRAS, and TP53 Mutations in Non-Small Cell Lung Cancers. Lung Cancer 2010, 69, 279–283.
  13. Sos, M.L.; Koker, M.; Weir, B.A.; Heynck, S.; Rabinovsky, R.; Zander, T.; Seeger, J.M.; Weiss, J.; Fischer, F.; Frommolt, P.; et al. PTEN Loss Contributes to Erlotinib Resistance in EGFR-Mutant Lung Cancer by Activation of Akt and EGFR. Cancer Res. 2009, 69, 3256–3261.
  14. Yoo, S.B.; Xu, X.; Lee, H.J.; Jheon, S.; Lee, C.T.; Choe, G.; Chung, J.H. Loss of PTEN Expression Is an Independent Poor Prognostic Factor in Non-Small Cell Lung Cancer. J. Pathol. Transl. Med. 2011, 45, 329–335.
  15. Cumberbatch, M.; Tang, X.; Beran, G.; Eckersley, S.; Wang, X.; Ellston, R.P.A.; Dearden, S.; Cosulich, S.; Smith, P.D.; Behrens, C.; et al. Identification of a Subset of Human Non-Small Cell Lung Cancer Patients with High PI3Kβ and Low PTEN Expression, More Prevalent in Squamous Cell Carcinoma. Clin. Cancer Res. 2014, 20, 595–603.
  16. Panagiotou, I.; Tsiambas, E.; Lazaris, A.C.; Kavantzas, N.; Konstantinou, M.; Kalkandi, P.; Ragkos, V.; Metaxas, G.E.; Roukas, D.K.; Vilaras, G.; et al. PTEN Expression in Non Small Cell Lung Carcinoma Based on Digitized Image Analysis. J. BUON 2012, 17, 719–723.
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