Hypoxic Microenvironment-Induced Reduction in PTEN-L Secretion Promotes Non-Small Cell Lung Cancer Metastasis through PI3K/AKT Pathway

Objective Lung cancer is the leading cause of cancer-related deaths worldwide. The aim of this study was to investigate the effects of hypoxic microenvironment on PTEN-L secretion and the effects of PTEN-L on the metastasis of non-small cell lung cancer (NSCLC) and the potential mechanisms. Methods The expression levels of PTEN-L in NSCLC tissues, cells, and cell culture media were detected. The transfection of PTEN-L overexpression construct or HIF-1α-siRNAs was conducted to manipulate the expression of PTEN-L or HIF-1α. NSCLC cells were introduced into 200 μM CoCl2 medium for 72 hours under 37°C to simulate hypoxia. The proliferation and apoptosis of the A549 cells were determined by the Cell Counting Kit-8 assay and Annexin V-FITC/PI-stained flow cytometry assay, respectively. Wound healing assay and transwell invasion assay were used to measure the migration and invasion of A549 cells. The protein expression of PTEN, PTEN-L, PI3K/AKT pathway-related proteins, and HIF-1α was detected by Western blot. Results PTEN and PTEN-L are downregulated in lung cancer tissues and cells. The protein expression of PTEN-L in the culture medium of lung cancer cell lines is decreased. The hypoxic microenvironment inhibits PTEN-L secretion. The low level of PTEN-L promotes cell proliferation, migration, and invasion, as well as inhibits apoptosis of A549 cells. The overexpression of PTEN-L attenuated the activation of the PI3K/AKT pathway by the hypoxic microenvironment. The knockdown of HIF-1α upregulates PTEN-L secretion under hypoxia. Conclusions The hypoxic microenvironment inhibits PTEN-L secretion and thus activates PI3K/AKT pathway to induce proliferation, migration, and invasion promotion, and apoptosis inhibition in NSCLC cells.


Introduction
Lung cancer is one of the most dreaded malignant tumors and the leading cause of cancer-related deaths across the world (approximately a quarter of cancer deaths) [1,2]. Non-small cell lung cancer (NSCLC) is the most commonly reported subtypes of lung cancer, which represents 85% of all lung cancer [3]. e strong invasive and metastatic nature of NSCLC leads to poor prognosis [4]. Despite developments in conventional (chemo)radiotherapy and surgery, the survival of NSCLC patients remains poor [5]. us, it is an increasingly urgent need to illuminate the molecular mechanisms underlying and new effective therapeutic strategies of NSCLC. e TME represents a dynamic cellular milieu in which the tumor exists [6,7]. TME composition differs by cancer types, yet signature characteristics comprise immune cells, stromal cells, blood vessels, and extracellular matrix. Hypoxia is present in most tumors and is caused by an imbalance between high oxygen consumption and insufficient oxygen delivery capacity [8]. Hypoxia can trigger cell death through apoptosis/necrosis; however, what it can also do is protect against cell death via stimulating adaptive responses, which in reverse promotes cell proliferation or angiogenesis, thereby promoting tumor development [9]. Transcription factor hypoxia-inducible factor-1α (HIF-1α) has an essential effect on cancer cellular metabolism. HIF-1α promotes glycolysis and facilitates tumor progression [10]. Nevertheless, the underlying molecular mechanism involved in the effect of tumor growth and metastasis remains unclear.
PTEN (phosphatase and tensin homolog deleted on chromosome 10), a prominent tumor suppressor gene [11,12], inhibits PI3K/AKT pathway via lipid phosphatase activity [13]. PTEN has a translation variant named PTEN-Long (PTEN-L) [14,15]. PTEN-L consists of all the domains of PTEN with the added 173 N-terminal amino acids in its N-terminal alternatively translated region (ATR) [14]. In many diseases, hypoxia affects PTEN secretion and thus regulates the progression of diseases. In nonalcoholic fatty liver disease, hypoxia induces HIF-1α accumulation, and consequently, PTEN expression is reduced, exacerbating liver fibrosis in nonalcoholic fatty liver disease [16]. In neonatal hypoxic-ischemic brain damage, HIF-1α inhibition of PTEN mediates the protective function of BMSCs on neurons under hypoxia [17]. In brain ischemia, reciprocally opposed binding partners of PTEN-L or PTEN in cytosolic or nuclear components are regulated following ischemic-like stress induced by oxygen-glucose deprivation [18]. PTEN has an important effect on various tumors. Sementino et al. [19] suggested that reduced Tp53 and PTEN activity in mouse mesothelium cells promotes mesothelioma progression. Shi et al. [20] exhibited that miR-29a reduces proliferation and drug resistance of colon cancer cells through upregulation of PTEN. Yu et al. [21] discovered that PTEN promoted NSCLC metastasis via the integrin αVβ6 pathway. Purified PTEN-L is taken up by tumor cells and regulates the PI3K/AKT pathway [22]. In vitro experiments also show that secreted PTEN-L inhibits the proliferation of U87 cells [23]. Based on these reports, we speculate that hypoxia may affect the metastasis of NSCLC via regulating the secretion of PTEN-L. e effect of PTEN-L on NSCLC cells remains unclear.
In our current research, we explored the role of hypoxic environments played in PTEN-L secretion and whether it has an impact on the progression of NSCLC. Understanding the effects of PTEN-L as an exogenous therapeutic agent in NSCLC contributes to the improvement of NSCLC.

Clinical Samples.
e NSCLC tissues and paired normal paracancerous tissues were derived from tissues derived from 30 NSCLC patients undergoing surgical resection. en, the samples were stored in a refrigerator at −80°C in time for subsequent experiments. All participants in this research were not receiving treatment prior to surgery. is study has been reviewed and approved by the Ethics Committee of the General Hospital of Ningxia Medical University Hospital.

Cell Culture and Treatment.
e human bronchial epithelial cell line (16HBE) and the human NSCLC cell lines A549, A549, H226, and H460 were maintained in RPMI 1640 medium. e human NSCLC cell line SPC-A1 was cultivated in Dulbecco's modified Eagle medium (Gibco, USA). All media consisted of 10% fetal bovine serum (FBS) and antibiotics. All cell cultures were preserved in a humidified atmosphere at 37°C with 5% CO 2 . All these cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). e protein expression levels of PTEN and PTEN-L in each cell and the level of PTEN-L secreted in the culture medium were detected.
To preliminarily analyze the effect of PTEN-L on NSCLC cells under the hypoxic microenvironment, A549 cells were divided into 4 groups: control group, hypoxia group, PTEN-L group, and hypoxia + PTEN-L group. e biological behavior of cells in each group was detected, and the changes in PI3K/AKT pathway in cells of each group were compared.
To analyze the effect of HIF-1α on the expression of PTEN-L under hypoxia, a rescue experiment was performed. A549 cells were divided into 4 groups: control, hypoxia, si-HIF-1α, and hypoxia + si-HIF-1α. e expression and secretion levels of PTEN-L in each group of cells were detected. e control group cells were cultured under the above conditions. In vitro hypoxic microenvironment was induced by CoCl2. PTEN-L was overexpressed, and HIF-1α was silenced by transfection. e specific experimental methods were as follows.

Plasmid Constructs.
e complementary DNAs (cDNAs) of PTEN-L were shown to be synthesized by reverse transcription and amplified using polymerase chain reaction (PCR). en, the cDNA has been incorporated into pCMV-Tag2B vector (N-terminal Flag tag). e constructs of Flag-PTEN-L were generated by subcloning. All DNA sequencing was used to confirm plasmid constructs. e expression of plasmid was confirmed by Western blot.

Cell Transfection.
One day prior to transfection, A549 cells were spread in a 24-well plate at a cell density of 1 × 10 5 cells/well. Cells were transfected with plasmid or siRNA when they reached 80% confluence. We performed the transfection using Lipofectamine 3000 reagent (Invitrogen) according to the instructions, and the transfected cells were incubated in a cell culture incubator at 37°C for 48 hours.  Assay. A549 cells with a density of 4 × 10 3 cells/well were spread in a 96-well culture plate. 10 μL CCK-8 (ImmunoWay Biotechnology Company) was added per 100 μL medium and then cultured for 4 h at 37°C. e absorbance at 450 nm was measured by a microplate reader.

Annexin V-FITC/PI-Stained Flow Cytometry Assay.
A549 cells were centrifuged (2000 rpm, 5 min) and resuspended (400 μL binding buffer). en, 5 ul of each Annexin V-FITC and PI was placed into the cell suspension. e cells and reagents are mixed and then left to stand (on ice, 10 min). Kaluza Analysis Software (Beckman Coulter, Inc.) was adopted to process the data of flow cytometry. e second quadrant and the fourth quadrant represent apoptotic cells.

Wound
Healing Assay. We dispersed A549 cells into a 6well plate (1 × 10 5 /well). A wound was generated by scratching the cell monolayer using a 200 μl pipette gun tip when the A549 cells reached near 100% confluence. Cell fragments were taken off by PBS. e plates were imaged, followed by the addition of the serum-free medium, and the plates were imaged again by incubation at 37°C for 24 hours. e wound healing processes were observed under a light microscope (magnification, ×100; Olympus Corporation) at 0 and 48 h after the scratch, and the distance was evaluated with ImageJ software. e experiment was conducted independently in triplicate.

Transwell
Assay. Cells were spread into the upper chamber of transwell plates with A549 (1 × 10 5 cells per well) and cultured with serum-free medium. Serum-free medium containing cells was added to the basement membrane matrix-coated upper chamber. e lower chamber was filled with media supplemented containing 10% FBS. e transwell chambers were kept in the incubator for 24 h, and cotton swabs were employed for wiping the cells remaining on the membrane upper surface. e cells on the transwell membranes were fixed with paraformaldehyde (4%) for 10 min. e crystal violet solution (0.5%) was used to stain. A microscope with a magnification of 100 (Olympus) was taken to count the number of invading cells.

Statistical Analysis.
e data were analyzed by SPSS software 23.0. Descriptive statistics were presented as the means ± SD. e method of comparison of quantitative variables between two groups was an unpaired t-test. Comparisons of qualitative variables between multiple groups were done by the one-way ANOVA. P < 0.05 was considered to be a significant difference.

Hypoxic Microenvironment Inhibits PTEN-L Secretion to Promote Metastasis of NSCLC and Activate PI3K/AKT
Signaling. Furthermore, we validated the effect of the decreased PTEN-L secretion caused by hypoxic microenvironment on the migration and invasion of A549 cells. e wound healing assay indicated that the wound healing percentage of A549 cells was elevated in the hypoxic group and decreased in the PTEN-L group in contrast to the control group. e hypoxia-mediated increase in wound healing rate in A549 cells can be inhibited by the upregulation of PTEN-L (Figure 3

Silencing HIF-1α Partially Reverses the Decrease in PTEN-L Secretion Induced by Hypoxic Environment.
Finally, we determined the effect of HIF-1α on PTEN-L and PTEN secretion under hypoxic environments in A549 cells. We found that HIF-1α expression was upregulated, whereas PTEN-L and PTEN-L expression was downregulated in hypoxic environments (Figures 4(a), 4(b), p < 0.01, p < 0.001). To determine whether HIF-1α regulates PTEN-L secretion under hypoxia, we knocked down HIF-1α (Figures 4(c), 4(d), p < 0.001). e results showed that the knockdown of HIF-1α can partially downregulate the elevation of PTEN and PTEN-L caused by hypoxia (Figures 3(e), 3(f ), p < 0.01, p < 0.001). Similarly, when compared to the hypoxia group, the knockdown of HIF-1α also significantly reversed the decrease in PTEN-L protein in the medium of the A549 cells (Figures 3(g), 3(h), p < 0.01, p < 0.001). e above findings revealed that the knockdown of HIF-1α promotes PTEN-L secretion under hypoxia. is suggested that HIF-1α was a key factor regulating the expression of PTEN-L under the hypoxic microenvironment.

Discussion
Sanctuary of the devil tumor cells constantly interacts with the surrounding microenvironment [6]. e tumor microenvironment affects cancer characteristics, such as its ability to promote proliferation and angiogenesis and inhibit apoptosis and the immune system [26]. e glycolysis in cervical cancer can be facilitated by CNPY2 under anoxic conditions [27]. e expression of mmu_circ_0000826 was raised under hypoxia, which in turn facilitates the formation and metastatic ability of colorectal cancer [28]. Hypoxia is a key feature of solid tumors. Hypoxia induces the downregulation of FAM13A and thus reduces the proliferation and metastasis of NSCLC [29].
PTEN has been reported to be carcinogenic, and a number of biological processes such as cell proliferation, growth, migration, metabolism, and death are regulated by PTEN [30]. Many studies have demonstrated that PTEN can be secreted in extracellular vesicles of exosomes [31]. It can also be secreted as a naked protein (longer heterodimer) [31]. e second form secreted is called PTEN-Long [31]. Similar to PTEN, PTEN-Long can inhibit the PI3K-AKT pathway and restrains cellular proliferation [32].
One investigation found that PTEN expression was reduced under continuous hypoxic stimulation [33]. Combined treatment of pancreatic cancer with GAS1 and PTEN inhibited cell invasion promoting cell death [34]. e reduced expression of Tp53 and PTEN promotes the progression of pleural and peritoneal malignant mesotheliomas [19]. miR-29a targets P-gp downstream of PTEN to induce drug resistance, proliferation inhibition, and apoptosis promotion in colon cancer cells [20]. In this study, we found that PTEN and PTEN-L were downregulated in NSCLC tissues and cells and PTEN-L was downregulated in the Evidence-Based Complementary and Alternative Medicine 5 medium of NSCLC cells. e finding that PTEN-L level is increased in paraneoplastic tissue cells backs up the results in our study [22]. e above findings indicate that PTEN and PTEN-L are likely to be involved in the development of NSCLC under hypoxic conditions. To verify whether PTEN and PTEN-L under hypoxia are involved in the development of NSCLC, we examined cell proliferation, apoptosis, migration, and invasion of A549 cells. We found that the hypoxic microenvironment inhibits PTEN-L secretion to induce proliferation, migration, and invasion promotion, and apoptosis inhibition in NSCLC cells. e study found that lncRNA ZEB2-AS1 promotes the proliferation, migration, and invasion of NSCLC cells through downregulating PTEN [35]. PTEN inactivation contributes to metastasis of NSCLC [36]. PTEN facilitates metastasis of NSCLC via the integrin αVβ6 pathway [21]. e intracellular signal transduction pathway PI3K/AKT is frequently overactivated in human cancers [37]. e PI3K/ AKT pathway is related to the development and progression of a range of cancers [38][39][40]. Zhang et al. [41] discovered that PI3K/AKT is involved in cisplatin resistance in NSCLC.
is suggests that hypoxia-mediated reduction in PTEN-L secretion is likely to mediate receptor tumor cell proliferation, metastasis, and apoptosis through activation of the PI3K/AKT pathway. A number of researches have reported the involvement of hypoxia-activated PI3K/AKT pathway in cancer progression. In colorectal cancer, hypoxia increases Nur77 expression to further activate PI3K/AKT pathway to induce EMT [48]. In glioma,  Evidence-Based Complementary and Alternative Medicine the expression of PLOD2 is increased under hypoxia, which can promote tumor proliferation and metastasis through PI3K/AKT signaling [49]. In hepatocellular carcinoma, hypoxia promotes TUFT1 expression, which activates the Ca 2+ /PI3K/AKT pathway to promote cancer growth and metastasis [50].
Hypoxia plays a driving role in the development of tumors [49]. It has been reported by many studies that hypoxia-inducible factor-1α (HIF-1α) induces hypoxia and regulates tumor cell adaptation to hypoxia in response to changes in oxygen [51]. Stability and levels of HIF-1α in cancer cells are elevated in hypoxic environments [52]. e study found that hypoxia/HIF-1α suppresses antagonistic tumor immune responses and promotes malignant tumor development [53]. erefore, we speculate that it is likely that HIF-1α is participated in the secretion of PTEN-L in the hypoxic microenvironment. Our results show that silencing HIF-1α can reverse the decrease in PTEN-L expression caused by hypoxia, suggesting that decreasing the level of HIF-1α promotes the secretion of PTEN-L under hypoxia. is suggests that cells in the hypoxic center of tumor tissue may regulate tumor cell proliferation and migration in normoxic environment by expressing PTEN-L. However, only cell experiments were performed in this study. e clinical value of PTEN-L in NSCLC deserves further analysis.
e effect of secreted PTEN-L on cell-to-cell communication in NSCLC needs to be confirmed by further cell and animal experiments.

Conclusion
Hypoxia induces a decrease in PTEN-L secretion in NSCLC. Hypoxia-induced reduction in PTEN-L induces proliferation and metastasis promotion, and apoptosis inhibition in NSCLC.
e PI3K/AKT pathway may be involved in the regulation of PTEN-L. Our study offers new insights into the role of PTEN-L on NSCLC development in response to hypoxic stimulation, which shows that PTEN-L is a new potential target for the treatment of NSCLC.
Data Availability e datasets used or analyzed during this study are available from the corresponding author on reasonable request.

Ethical Approval
is study has been approved by our hospital's ethics committee in compliance with the Declaration of Helsinki. All patients volunteered to join this study. e written informed consent has been signed by all participants.

Disclosure
Xuyang Song and Jinxi He are co-first authors.

Conflicts of Interest
All authors declare that they have no conflicts of interest.