Alkaline phosphatase downregulation promotes lung adenocarcinoma metastasis via the c-Myc/RhoA axis

Background Lung adenocarcinoma (LUAD) metastasis significantly reduces patient survival; hence inhibiting the metastatic ability of lung cancer cells will greatly prolong patient survival. Alkaline phosphatase (ALPL), a homodimeric cell surface phosphohydrolase, is reported to play a controversial role in prostate cancer and ovarian cancer cell migration; however, the function of ALPL in LUAD and the related mechanisms remain unclear. Methods TCGA database was used to analysis the expression of ALPL, and further verification was performed in a cohort of 36 LUAD samples by qPCR and western blot. Soft-agar assay, transwell assay and lung metastasis assay were employed to detect the function of ALPL in LUAD progression. The qPCR, luciferase promoter reporter assay and western blot were used to clarify the molecular mechanisms of ALPL in promoting metastasis in LUAD. Results ALPL was downregulated in LUAD, and the disease-free survival rate of patients with low ALPL was significantly reduced. Further studies showed that overexpression of ALPL in LUAD cell lines did not significantly affect cell proliferation, but it did significantly attenuate lung metastasis in a mouse model. ALPL downregulation in LUAD led to a decrease in the amount of phosphorylated (p)-ERK. Because p-ERK promotes the classical c-Myc degradation pathway, the decrease in p-ERK led to the accumulation of c-Myc and therefore to an increase in RhoA transcription, which enhanced LUAD cell metastasis. Conclusion ALPL specially inhibits the metastasis of LUAD cells by affecting the p-ERK/c-Myc/RhoA axis, providing a theoretical basis for the targeted therapy of clinical LUAD. Supplementary Information The online version contains supplementary material available at 10.1186/s12935-021-01919-7.

the main pathological type of lung cancer [3]. Clinically, more than 60% of patients present with later-stage (stage III or IV) disease, which contributes to the poor prognosis of LUAD [3]. Furthermore, the data for [2007][2008][2009][2010][2011][2012][2013] in the USA showed that the 5 year survival rate for lung cancer decreases from 56 to 29% to 5% as the disease progresses from localized to regional and then to distant, respectively [4], indicating that lung cancer metastasis significantly reduces patient survival. Therefore, inhibiting the metastatic ability of lung cancer cells will greatly prolong patient survival.
Studies of the gene encoding alkaline phosphatase (ALPL) are focused mainly on hypophosphatemia. Various mutations in this gene have been shown to affect its activity and cause a decrease in serum ALPL levels, resulting in hypercalcemia and hypokalemia [5]. ALPL plays a crucial role in intracellular signaling and regulation of protein activity [6][7][8]. Dysfunction of this enzyme is related to the development of multiple diseases, including diabetes mellitus, liver disease, stork and Alzheimer's disease [9][10][11][12]. According to recent reports, ALPL also plays a role in cancer. Rao et al. found that knockdown of ALPL can reduce prostate cancer cell migration [10]. Another study suggested that overexpression of ALPL can inhibit the migration and invasion of HGSOC ovarian cancer cells [13]. Therefore, the function of ALPL in cancer and the related mechanisms remain controversial.
c-Myc is one of the most well-characterized transcription factors in tumors. Analysis of a hybrid of rodent and human Burkitt lymphoma cells revealed the presence of a reciprocal translocation between chromosomes 8 and 14 that disrupted the MYC gene [14], which confirmed that MYC functions as an oncogene in humans [15]. Translocations in MYC occur often in hematopoietic cancers, and it was reported to be the third most frequently amplified gene in human cancers in a whole-genome copy number analysis [16,17]. Its downstream effector, RhoA, is a member of the Rho family of small GTPases and is a key regulator of actin polymerization and cell migration [18,19]. It acts as a molecular switch in cellular processes such as cell morphogenesis, adhesion, and migration [20,21]. The primary function of RhoA is to remodel the actin cytoskeleton during cellular processes such as migration and endocytosis, which also affects the local dynamics of microtubules (MTs) [22]. Thus, RhoA clearly plays an important role in cell migration and invasion [23,24].
In this study, we revealed a molecular mechanism through which ALPL regulates LUAD invasion and metastasis by affecting the c-Myc/RhoA cascade. These findings provide a theoretical basis for the targeted therapy of clinical LUAD.

Reverse transcription-PCR
Total RNA was extracted using TRIzol reagent (Invitrogen/ThermoFisher Scientific) according to the manufacturer's instructions. Next, 5 μg total RNA was used for first-strand cDNA synthesis with the SuperScript First-Strand Synthesis system and oligo (dT) primers (Invitrogen), as described previously [28].

Soft-agar assay
The anchorage-independent growth ability of the cells was evaluated in soft agar, as described [30]. Briefly, 3 mL of 0.5% agar (214010; Becton Dickinson) in basal modified Eagle's medium supplemented with 10% FBS was layered onto each well of 6-well tissue culture plates. Cells (3 × 10 4 ) suspended in 1 mL normal medium (B9638; Sigma-Aldrich) were mixed with 2 mL of 0.5% agar in basal modified Eagle's medium supplemented with 10% FBS, and 1 mL of the mixture was added into each well on top of the 0.5% agar layer. Plates were incubated at 37 °C in 5% CO 2 for 3 weeks, and the colonies were counted. The data is presented as the number of colonies/10 4 cells [30].

Cell migration and invasion assay
Cell migration and invasion were evaluated using kits from BD Falcon. The assay was performed according to the manufacturer's instructions, as described previously [27,31]. Briefly, cells were seeded in the inserts in 400 μL serum-free medium. The inserts were placed in wells containing 700 μL medium with 10% FBS and incubated for 24 h. Then, cells on the upper surface of the filters were removed with a cotton swab. The inserts were fixed in methanol and stained with Giemsa, imaged with a microscope, and then the cells were counted. The data shown are representative of three independent experiments [32].

Tube formation assay
Tube formation assay was conducted on HUVECs under the treatment of different LUAD cancer cell culture medium. HUVECs (2.0 × 10 4 cells) were cultured in the presence of the conditioned medium in 96-well plates coated with Matrigel (50 μL/well, BD Biosciences). After 4 h incubation at 37 °C, tube formation was visualized under light microscope. Capillary length was calculated with Image J software. This assay was carried out in triplicate.

Luciferase promoter reporter assay
The RhoA luciferase reporter and pRL-TK were each transiently co-transfected into A549 and HCC827 cells. Luciferase activity was determined 24 h after transfection using the Luciferase Assay System (Promega, Madison, WI, USA), as described [27,33,34]. The values were normalized to the internal thymidine kinase (TK) signal. All experiments were conducted in triplicate, and the results were expressed as the mean ± standard error [27].

H&E staining
Fixed lung tissues were dehydrated using an alcohol gradient, paraffin-embedded, and then stained with H&E, as described in detail previously [35]. Images were captured using the Nikon Eclipse Ni microsystem (Nikon DS-Ri2; Tokyo, Japan).

Immunohistochemistry (IHC)
IHC assays were performed to detect the expression of ALPL, p-ERK, c-Myc and RhoA in formalin-fixed paraffin-embedded metastatic lung tumor tissues of mice injected with LUAD cells. Specific antibodies against ALPL (GTTX100817, Genetex), p-ERK1/2 (4370S, CST), c-Myc (sc-764, Santa Cruz) and RhoA (sc-418, Santa Cruz) were used for IHC staining, which were performed using a kit from Boster Bio-Engineering Company (SA1022; Wuhan, China). Immunostained images were captured with the Nikon Eclipse Ni microsystem (DS-Ri2) and analyzed with Image-Pro Plus version 6.0 (Media Cybernetics, Rockville, MD, USA) by calculating the integrated optical density (IOD) of each stained area (IOD/area). At least five images per specimen were counted.

Clinical specimens
In total, 36 pairs of human LUAD tissues and corresponding adjacent normal tissues were obtained. The authorized case information is shown in Additional file 1: Table S1. Preoperative biopsy and postoperative pathological examination of the above patients confirmed LUAD. The tissue samples were snap-frozen in liquid nitrogen at the time of surgery, RNA was extracted, and cDNA was synthesized and stored at − 80 °C.

Statistical analysis
All experimental data are expressed as the mean ± standard deviation (mean ± SD). Graphs and statistical analyses were performed using GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA). The Kaplan-Meier method was used to draw the survival curve, and the logrank test was used to compare the differences between groups. Comparisons between the control group and the experimental group were performed using the Student's t-test. Correlation between two independent groups were performed using the Pearson's Chi-square test. P < 0.05 was considered statistically significant. c Western blot analysis of total protein lysates prepared from 36 paired fresh clinical LUAD tissues. Then perform grayscale analysis and statistics. d The relationship between ALPL expression in LUAD patients in the TCGA database and the disease-free survival rate. e Western blot analysis of ALPL protein levels in LUAD cell lines. GAPDH was used as an internal control. All results were presented as the mean ± SD and analyzed by Student's t-test. The survival curve was analyzed by log-rank test. Asterisks (*) represent statistical significance (P < 0.05)

ALPL is downregulated in LUAD and related to patient prognosis
Analysis of the 58 paired LUAD cases in the Cancer Genome Atlas (TCGA) database revealed significantly lower expression of ALPL mRNA in LAUD tumors compared with matched healthy lung tissues (Fig. 1a). A separate qPCR analysis of 36 fresh LUAD samples and matched healthy tissues revealed significantly lower ALPL expression in tumor tissues, consistent with the results obtained using the TCGA database (Fig. 1b). Furthermore, western blot experiments were performed on 36 paired LUAD samples, and grayscale analysis was used to make statistics. The results showed that compared with matched healthy tissues, the expression of ALPL protein in LUAD tumors was significantly down-regulated (Fig. 1c, Additional file 1: Fig. S1). Most important, a survival analysis of LUAD patients in the TCGA database revealed that patients with low expression of ALPL had a significantly worse prognosis (Fig. 1d). We therefore concluded that ALPL downregulation in LUAD might be an important molecular event for cancer development or progression. Then we examined the protein expression of ALPL in normal lung cells and LUAD cells, and the results showed that the expression of ALPL in LUAD cells was lower than that in normal lung cells (Fig. 1e).

ALPL has no effect on LUAD cell proliferation
To investigate the function of ALPL in LUAD cells, we selected two LUAD cell lines, A549 and HCC827, which have relatively low expression of ALPL, and established cell lines stably overexpressing ALPL (Fig. 2a, b). A human lung squamous cell carcinoma (LUSC) cell line (SK-MES-1) stably overexpressing ALPL was also generated (Additional file 1: Fig. S2a). To assess the effects of ALPL on LUAD cell growth, soft-agar experiments were carried out. ALPL overexpression had no effect on the anchorage-independent growth of LUAD ( Fig. 2c-f ) or LUSC cells (Additional file 1: Fig. S2b, c).

ALPL significantly inhibits the migration and invasion of LUAD cells, and represses LUAD metastasis in a xenograft model
The prognosis of lung cancer is significantly worse once it has metastasized. Evaluation of the TCGA database revealed higher levels of ALPL in patients with stage I LAUD compared with patients with later-stage disease (II, III, and IV; Additional file 1: Fig. S3). This suggested that ALPL expression may be closely related to LUAD metastasis. To evaluate this, we performed Transwell and wound healing experiments to investigate the effects of ALPL on LUAD cell migration and invasion. ALPL overexpression significantly inhibited the migration and invasion of both A549 and HCC827 cells (Fig. 3a-d, Additional file 1: Fig. S4). By contrast, ALPL overexpression had no effect on the migration or invasion of LUSC cells (SK-MES-1; Additional file 1: Fig. S2d, e). This result may explain the observation that the survival of LUSC patients in the TCGA database was not related to ALPL expression (Additional file 1: Fig. S5). The effect of ALPL on the metastatic capacity of LUAD cells was verified in vivo in a nude mouse xenograft model of lung cancer metastasis. ALPL overexpression significantly reduced the number and size of A549 lung cancer metastases ( Fig. 3e-g, Additional file 1: Fig. S6). ALPL overexpression had the same effect in HCC827 cells (Additional file 1: Fig. S7a-c). Moreover, the results of tube formation assay showed that ALPL overexpression also inhibited angiogenesis (Fig. 3h, i). Therefore, in both in vivo and in vitro experiments, ALPL overexpression specially inhibited the migration of LUAD cells.

ALPL inhibits LUAD cell migration by downregulating RhoA
To elucidate the molecular mechanism through which ALPL inhibits LUAD cell migration and invasion, western blot assays were performed to evaluate the expression of molecules related to cell motility. Matrix metalloproteinase (MMP)2 and MMP9 enhance the invasion of cancer cells by degrading type IV collagen [36,37]. Slug, Snail, E-cadherin, N-cadherin, and β-catenin, which are involved in epithelial-mesenchymal transition (EMT), have also been reported to promote cancer cell migration and invasion [17,18,[38][39][40][41][42][43], while RhoA, Cdc42, and Rac1 are members of the Rho family of small GTPases, and are key mediators of actin polymerization and cell migration. These molecules play active roles in cell migration and invasion [22,44,45]. RhoA was consistently and significantly downregulated in A549 and HCC827 cells after overexpression of ALPL, while the other molecules were not consistently affected by ALPL expression (Fig. 4a). Therefore, we hypothesized that ALPL inhibited LUAD cell migration by downregulating RhoA expression. To validate this hypothesis, RhoA was stably overexpressed in the ALPL-overexpressing cell lines [A549 (ALPL) and HCC827 (ALPL)], as shown in Fig. 4b and e. Overexpression of RhoA restored the migratory ability of A549 and HCC827 transfectants (Fig. 4c, d and f, g). These findings suggest that ALPL inhibits LUAD cell migration by downregulating RhoA.

ALPL inhibits RhoA transcription by downregulating c-Myc
To further explore the mechanism by which ALPL downregulates RhoA expression, we first conducted a semiquantitative PCR analysis of the RhoA mRNA level. RhoA mRNA was significantly downregulated in A549 (ALPL) and HCC827 (ALPL) cells by PCR (Fig. 5a) and qPCR (Fig. 5b). These data indicated that ALPL downregulated RhoA expression at the mRNA level. To investigate the mechanism underlying ALPL downregulation of RhoA, we investigated RhoA promoter activity by dual-luciferase reporter assays. As shown in Fig. 5c, RhoA promoter activity was significantly downregulated in both cell lines after ALPL overexpression, indicating that ALPL downregulates RhoA expression by inhibiting its transcription.
To further investigate the effects of ALPL on RhoA transcription, the TFANSFAC Transcription Factor Binding Sites Software (Biological Database, Wolfenbuttel, Germany) was used to predict transcription factors controlling RhoA expression. Transcription factors for which potentially conserved binding sites in the RhoA promoter region were identified included c-Myc, c-Jun, Foxp3, and Sp1 (Fig. 5d). We examined the expression of these potential transcription factors in A549 and HCC827 cells by western blot analysis. c-Myc protein levels were significantly downregulated in A549 (ALPL) and HCC827 (ALPL) cells, while the other molecules showed no signs of consistent downregulation (Fig. 5e). It has been reported that c-Myc directly regulates RhoA transcription [44]. We hypothesized that c-Myc may be a key molecule in the mechanism through which ALPL regulates RhoA transcription. To test this hypothesis, we constructed an A549 (ALPL) stable cell line that expresses c-Myc and found that RhoA protein expression was restored (Fig. 5f ). Subsequent qPCR analysis confirmed that RhoA expression was also restored at the mRNA level (Fig. 5g). Dual-luciferase reporter assays also showed that overexpression of c-Myc restored the level of RhoA promoter activity in A549 (ALPL) cells (Fig. 5h). These data suggested that ALPL-downregulated c-Myc expression in LUAD resulted in reduced RhoA expression, which in turn resulted in decreased LUAD cell The migration and invasion ability of A549 (ALPL) cells was restored following transfection with c-Myc (Fig. 5i,  j). These data confirm that ALPL downregulates RhoA by inhibiting the expression of c-Myc, resulting in reduced LUAD cell migration and invasion.

ALPL promotes the degradation of c-Myc by activating ERK
To further explore the mechanism by which ALPL regulates c-Myc, we analyzed c-Myc mRNA levels by qPCR. There were no significant differences in c-Myc mRNA levels in A549 (ALPL) and HCC827 (ALPL) cells compared with the corresponding wild-type cell lines (Fig. 6a), indicating that ALPL does not regulate c-Myc at the transcriptional level. A variety of proteins have been reported to play roles in cancer development by regulating the degradation of c-Myc protein [45,46]. Therefore, we evaluated c-Myc protein degradation in control A549 and A549 (ALPL) cells. ALPL overexpression significantly enhanced the degradation rate of c-Myc protein (Fig. 6b), indicating that ALPL downregulates c-Myc expression by enhancing its degradation. It has been reported that degradation of c-Myc usually occurs via classical ubiquitin-dependent degradation pathways, in which ERK-and GSK-3-mediated phosphorylation of c-Myc at Ser62 and Thr58 tags it for degradation [47,48]. To confirm that ALPL degradation of c-Myc is ubiquitin-dependent, we investigated the effects of the proteasome inhibitor MG132 on c-Myc levels and phosphorylation in control A549 and A549 (ALPL) cells. After treatment with MG132, c-Myc accumulated in both the control A549 and A549 (ALPL) cells, with no significant difference between the two cell lines. However, levels of p-c-Myc (Thr58/ Ser62) were much higher in A549 (ALPL) cells than in control cells (Fig. 6c). These results suggested that ALPL accelerated the degradation of c-Myc by promoting its phosphorylation at Thr58 and Ser62, resulting in ubiquitin-dependent degradation.

ALPL overexpression increases p-ERK accompanied with c-Myc and RhoA downregulation in metastatic lung tumor tissues of mouse model
To determine whether ALPL exerts a regulatory effect on p-ERK/c-Myc/RhoA in vivo, we assessed the expression of ALPL, p-ERK, c-Myc and RhoA using IHC in metastatic lung tumor tissues of mouse models that are generated with A549 (ALPL) and HCC827 (ALPL) in comparison to A549 (Vector) and HCC827 (Vector), respectively, and found that a remarkable upregulation of p-ERK but c-Myc and RhoA downregulation in metastatic lung tumor tissues of mouse models generated by injection of A549 (ALPL) and HCC827(ALPL) cells ( Fig. 7a-d, Additional file 1: Fig. S8a-d). RhoA is positively correlated with c-Myc expression, while c-Myc expression is negatively correlated with p-ERK expression. Collectively, these results demonstrated that ALPL expression downregulated c-Myc by enhancing ERK phosphorylation, leading to a decrease in c-Myc-mediated RhoA protein expression, which eventually suppresses LUAD cell metastasis (Fig. 8).

Discussion
In this study, we show for the first time that ALPL specially inhibits the metastasis of LUAD cells but not LUSC cells. This not only indicates that ALPL plays a unique role in the pathological development of LUAD, but also implicates ALPL as a specific target for inhibiting the metastasis of LUAD. We also show that the inhibitory effects of ALPL on the metastasis of LUAD cells are mediated via the p-ERK/c-Myc/RhoA axis. ERK, c-Myc and RhoA are the genes playing vital roles in tumor metastasis of many cancers including lung cancer. It is of great significance to develop drugs targeting ERK, c-Myc and RhoA for the treatment and prevention of tumor metastasis. Our results showed that ALPL can significantly inhibit the invasion of LUAD cells in vitro and in vivo, which is mediated via the ALPL/p-ERK/c-Myc/ RhoA axis. Therefore, our findings provide a theoretical basis for the development of strategies targeting ALPL for the treatment of LUAD.
ALPL is commonly used to evaluate acute liver damage and cholestasis [49]. Studies have also shown that ALPL is associated with hypophosphatemia [5,50], skeletal development, cardiovascular disease, and acute kidney injury [51][52][53]. It has been reported that mutations in ALPL affect its ability to cause disease [5]. We did not identify any mutations in ALPL in LUAD using the TCGA database, suggesting that metastasis of LUAD Fig. 7 The expression of ALPL, p-ERK, c-Myc and RhoA were determined by IHC staining in the metastatic lung tumors tissues. a Representative IHC images showing the expression of ALPL, p-ERK, c-Myc and RhoA in metastatic lung tumors tissues, n = 9, Scale bars: 400 µm or 100 µm. Protein expression levels of p-ERK (b), c-Myc (c), and RhoA (d) were analyzed by calculating the integrated optical density per stained area (IOD/area) using Image-Pro Plus version 6.0, and the correlation between p-ERK/c-Myc/RhoA and ALPL was analyzed by Pearson's Chi-square test cells is related to inhibition of ALPL expression rather than mutation. LUAD cells also had lower expression of ALPL than normal lung cells. ALPL also plays very different roles in different tumor types. Reduced expression of tumor-derived ALPL inhibited mesenchymal-to-epithelial transition and the migration of advanced prostate cancer [10]. Conversely, ALPL functions as a tumor suppressor gene in meningioma [54] and ovarian cancer [13]. In this study, we show for the first time that ALPL inhibits the metastasis of LUAD cells via the p-ERK1/2-c-Myc-RhoA axis, which extends our understanding of the role of ALPL in cancer development. Although the expression of ALPL is not tissue-specific, the function of ALPL is influenced by its cellular context. ALPL expression is elevated after acute injury of some organs such as the liver and kidneys [49,53]. Our finding that downregulation of ALPL promotes metastasis of LUAD cells highlights the diverse functions of ALPL in different settings. We further found that ALPL inhibited metastasis of LUAD cells but did not play a similar role in LUSC cells, and this phenomenon deserves further exploration.
In this study, we demonstrated that ALPL enhances ERK phosphorylation; however, previous studies have shown that ALPL downregulates its targets [6]. Lee et al. found that the human dental pulp cells were cultured in osteogenic medium, ALPL and p-ERK were up-regulated, while MKP-1 was downregulated [55]. As a member of the MAP kinase phosphatase (MKP) family, MKP-1 inactivates MAP kinases including ERK1/2 [56], and its degradation can be attenuated by inhibitors of the ubiquitin-directed proteasome complex. We speculate that ALPL may increase ERK phosphorylation by regulating MKP-1, although this remains to be clarified. ERK is a member of the MAPK family and promotes ubiquitindependent degradation of c-Myc by phosphorylation [47,48]. In our study, ALPL increased ERK phosphorylation, resulting in increased phosphorylation of c-Myc, thereby promoting c-Myc degradation. Furthermore, in LUAD cells, c-Myc degradation was reduced because p-ERK was reduced. Previous studies have shown that GSK3 plays a key role in the ubiquitin-dependent degradation of c-Myc [47,48]. However, our results indicated that p-ERK, not p-GSK3, is the key molecule through which ALPL promotes c-Myc ubiquitination. This finding led us to reexamine the status of p-ERK in the ubiquitin-dependent degradation of c-Myc.
c-Myc promotes RhoA mRNA expression, thereby enhancing the migration and invasion ability of tumor cells [44]. We demonstrate here that ALPL enhances the ubiquitin-dependent degradation of c-Myc by p-ERK. c-Myc is a well-known oncogene [15], and inhibition of c-Myc in cancer is a potential therapeutic strategy. Thus, the ability of ALPL to down-regulate c-Myc may represent a novel approach to cancer treatment. As a member of the Rho family, RhoA plays an important role in cancer cell metastasis [23,24]. Overexpression of RhoA significantly restored the inhibitory effect of ALPL on LUAD cells metastasis. Our study is the first to show that ALPL reduces the expression of RhoA to inhibit the metastasis of LUAD cells. In summary, ALPL promotes ubiquitin-dependent c-Myc degradation by activating ERK. Reduced c-Myc expression results in reduced RhoA transcription, thereby inhibiting cell migration and invasion. Accordingly, downregulation of ALPL decreased ERK phosphorylation in LUAD, causing an accumulation of c-Myc, which promotes RhoA transcription, leading to increased metastasis of LUAD cells (Fig. 8).

Conclusions
Overall, we evidenced for the first time that ALPL specially inhibits the metastasis of LUAD cells but not LUSC cells, and revealed a molecular mechanism through which ALPL regulates LUAD invasion and metastasis by affecting the p-ERK/c-Myc/RhoA axis. These results implicated ALPL as a specific target for inhibiting the metastasis of LUAD and provided a theoretical basis for the targeted therapy of clinical LUAD.

Supplementary Information
The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s12935-021-01919-7.  Fig. 8 Schematic diagram of the pathway by which ALPL downregulates RhoA via c-Myc. ① ALPL leads to an accumulation of phosphorylated (p)-ERK. ② p-ERK promotes c-Myc phosphorylation at Thr58 and Ser62 which is then degradated through ubiquitinproteasome system. ③ The reduction of c-Myc leads to a decrease in RhoA transcription, which eventually suppresses LUAD cell metastasis. The dotted line indicates a multi-process, and the solid line indicates a single process