BNIP3 enhances pancreatic cancer cell migration and proliferation via modulating autophagy under hypoxia

Chemotherapy and immunotherapy for pancreatic ductal adenocarcinoma (PDAC) have limited success for the intricated surrounding cancer microenvironment. Hypoxic microenvironment in PDAC causes the activation of multiple different molecules and signaling pathways compared with normoxia. We studied the roles of BNIP3 for the migration and proliferation of PDAC and Panc1 cells in vitro. In the present study, we found that BNIP3 expression was elevated and enhanced the migration and proliferation of CFPAC-1 and Panc1 cells under hypoxia. The upregulation of BNIP3 was important for the autophagic activation, while inhibition of autophagy with siRNA targeting Atg5 and Atg7 impaired the hypoxia-induced cell migration and proliferation. Additionally, blocking ERK1/2 mitogen-activated protein kinase (MAPK) signaling with PD98058 significantly down-regulated BNIP3 expression, autophagic activation, as well as the migration and proliferation of CFPAC-1 and Panc1 cells under hypoxia. Collectively, our results here uncover a hitherto unknown hypoxia-BNIP3-autophagy axis in modulating the migration and proliferation and provide a potential intriguing drug target for the therapy of PDAC.


Introduction
Pancreatic ductal adenocarcinoma (PDAC) remains an aggressive malignancy with a median survival about 6 months and the 5-year survival approximately 9% [1,2]. PDAC is considered to be the second leading cause of cancer deaths within the next decade due to the lack of improved outcomes. Its intractability is mainly resulted from the late diagnosis, early dissemination of metastases, a paucity of specific and sensitive biomarkers, as well as the resistance to chemotherapy, radiotherapy, and even immunotherapy, which is resulted from inherent intratumor heterogeneity and a highly desmoplastic and immunosuppressive tumor microenvironment (TME) [3,4].
Hypoxia, one of the most important TME, is a hallmark of many solid tumors that is related to the poor prognosis, treatment resistance, and elevated invasion and metastasis [5]. Hypoxia is clinically identified in the PDAC [6], and the investigation of orthotopic patient-derived xenografts (PDX) and genetically engineered mouse models (GEMM) indicates the correlation between hypoxia TME and the rapid tumor progression and metastasis. Intratumoral hypoxia leads to the upregulation of BNIP3 (Bcl-2 and adenovirus E1B 19-kDa interacting protein 3) [7], one of the known downstream targets of HIF1α (hypoxia-inducible factor 1 subunit alpha) [8]. BNIP3, a BH3-only protein that belongs to the Bcl-2 family, is proved with an emerging role in cell apoptosis and autophagy. Recent studies highlight a critical role for BNIP3 in carcinogenesis, such as laryngeal cancer, gastric cancer, glioma, melanoma, and hepatocellular carcinoma [9,10,11,12]. However, a considerable debate still remain underlying the cellular processes and mechanisms regulated by BNIP3 in cancer cells. Moreover, whether and how BNIP3 regulates the progression and dissemination of PDAC cell remains largely unclear.
Autophagy is one of the resistance mechanism in tumor therapy that promotes cell survival in adverse environments, although the uncontrolled autophagy can promote apoptosis [13]. There are three types of autophagy, i.e. macroautophagy, chaperone-mediated autophagy, and microautophagy. Macroautophagy (hereafter referred to as autophagy) is indicated as the main autophagy process activated when cells suffering from nutrient insufficiency and hypoxia [14]. Previous studies have shown that autophagy is activated when the pancreas is invaded by the intravascular pancreatic neoplasia [15]. Additionally, the PDAC cells have been demonstrated to rely on autophagy for nutrients and energy to survive in vitro [16]. A recent study suggests that autophagic flux can affect the immunogenicity of PDAC cells and that inhibiting autophagy can improve the sensitivity of PDAC cells to immunotherapy [17]. Thus autophagy acts a vital role for the occurrence, progession, and treatment.
Here, we set out to determine whether the BNIP3-induced autophagy impinges on the hypoxia response in PDAC cells. Indeed, our results show that BNIP3 up-regulates, mediates cell autophagy, and enhances the migration and proliferation of CFPAC-1 cells under hypoxia. In addition, we provide evidence that ERK1/2 MAPK signaling is significant activated and enhances BNIP3-mediated autophagy in CFPAC-1 cells under hypoxia. Inhibition of ERK1/2 signaling suppressed the migration, as well as the proliferation of CFPAC-1 cells under hypoxia. We conclude that hypoxic TME promotes the migration and proliferation of pancreatic cancer cell via modulating the BNIP3-autophagy axis with the involvement of ERK1/2 signaling pathway.

Hypoxia exposure
The Forma Series II Water Jacket CO 2 incubator (model: 3131; Thermo Scientific) was applied for the hypoxic conditions of 1% O 2 , 5% CO 2 and 93% N 2 , as well as the required temperature (37 C) and O 2 level with a constant flow of nitrogen. An ERK1/2 inhibitor PD98059 was purchased from MedChem Express (HY-12028, 10 μM) was added to the cells and incubated for 1 h prior to hypoxic treatment.

Scratched wound healing assay
Cell monolayers cultured in 6-well plates were scratched by a 100-μL plastic pipette tip after incubating for 2 h with mitomycin-C (S8146, Selleck, 5 μg/mL) to inhibit the proliferation. Healing process of the wound was monitored under an inverted light microscope (Olympus, Japan). The wound-closure rate (%) was measured by NIH ImageJ software (http://rsb.info.nih.gov/ij/), which indicated the potential of cell migration.

Single cell motility assay and quantitative analysis
The cells were cultured at a density of 0.5 Â 10 4 /cm 2 in 24-well plates. Then time-lapse imaging was conducted with a Zeiss imaging system (Carl Zeiss Meditec, Jena, Germany) in a temperature-and CO 2controlled chamber. The images were taken every 10 min for 3 h. The cells' trajectories were obtained using NIH ImageJ software by tracing the position of cell nucleus. The velocity of single cell movement (μm/ min) was defined as the accumulated distance (μm) of the trajectories dividing by time (min).

Cell proliferation assay
5-Ethynyl-2'-deoxyuridine (EdU), a thymidine analog, was incorporated to label cells undergoing DNA replication. EdU-positive rate of cells was applied to evaluate the proliferative ability, as determined by EdU staining with the Click-iT ™ EdU kit according to the manufacturer's instructions (BCK488-IV-IM-S, Sigma, USA) [18]. The Leica Confocal Microscope (SP5, Leica Microsystems, Germany) was used for capturing the fluorescent images. The Cell Counting Kit-8 (CCK-8, Genview, USA) assay was conducted refering to the manufacturer's instructions.

Lentivirus infection and small interfering RNA (siRNA) transfection
For the construction of stable cell line, BNIP3-overexpressing lentivirus (AdBNIP3) or the corresponding control lentivirus (AdNC) (Table S1) were obtained from the GeneChem Company (Shanghai, China) and used according to the manufacturer's instructions. The siR-NAs, purchased from GenePharma Company (Shanghai, China), were transfected into cells for the interfering of BNIP3, Atg5 or Atg7 (Table S1) with lipofectamine 2000 (11668027, Invitrogen) according to the manufacturer's protocol.

Immunoprecipitation (IP)
To test the interaction between BNIP3 and LC3, whole cell extracts were collected in the lysis buffer (Beyotime, P0013) and centrifuged at 14,000 g for 15 min. The supernatants were obtained and incubated with 2 μg of anti-LC3B (L7543, Sigma), anti-BNIP3 (ab10433, Abcam) at 4 C for 8 h, then precipitated with the Protein A/G-Sepharose (sc-2003, Santa Cruz Biotechnology) overnight at 4 C. The binding proteins were detected using western blotting. The denatured IgG light chain of the primary antibodies used for IP runs at about 25 kDa on the subsequent blotting, which may obscure the bands of BNIP3 (26 kDa). An secondary antibody specific for IgG heavy-chain was used to resolve this problem.

Recombinant adenovirus construction and transduction
The mRFP-GFP-LC3, Ad-Dsred-Mito (marking the mitochondrial) and Ad-GFP-LC3 (marking the autophagosome) adenovirus were obtained from the Hanbio Biotechnology (Shanghai, China) to detect autophagy flux and mitophagy, respectively. The cells were cultured on glass coverslips in 24-well plates and then transfected with these adenovirus overnight before the corresponding treatment. The fluorescent images were captured by a Leica Confocal Microscope (SP5, Leica Microsystems, Germany).

Statistical analysis
All these data were analyzed using SPSS statistics 22.0 software and presented as means AE SEM. Independent t test was used to explore the difference between two groups. One-way analysis of variance (ANOVA with Post Hoc LSD) were applied to evaluate the difference among three or more groups in all measured parameters. A p < 0.05 was considered as the statistical significance.

BNIP3 enhanced the migration and proliferation of pancreatic cancer cells under hypoxia
To evaluate the influence of hypoxia on the expression of BNIP3 in PDAC under hypoxic microenvironment, the CFPAC-1 cells were randomized into four groups and pre-subjected to hypoxia (1% O 2 ) for 12, 24 or 48 h before the detection of BNIP3 expression with the Western blot assay. Remarkably, hypoxia promoted BNIP3 expression in a timedependent manner, with the maximum effect achieved by hypoxia for 24 h ( Figure 1A and 1B). Meanwhile, the immunofluorescence staining showed a significant increase of BNIP3 expression in CFPAC-1 cells under hypoxia ( Figure 1C). Then the CFPAC-1 cells were transfected with BNIP3-specific siRNA (siBNIP3) or a scrambled sequence (siNC). As expected, BNIP3 was significantly downregulated with siBNIP3 transfection ( Figure 1D and 1E).
In order to explore the effects of BNIP3 on the migration and proliferation, CFPAC-1 cells transfected with siBNIP3 or siNC were subjected to normoxic or hypoxic condition. Western blot further comfirmed the knockdown of BNIP3 with siRNA transfection ( Figure 1F and 1G). As depicted in Figure 1H and 1I, a wound healing assay was performed to evaluate the role of BNIP3 for the cell migration. Results revealed that hypoxia promoted the migration of CFPAC-1 cells, while BNIP3 knockdown suppressed the cell migration under both normoxia and hypoxia condition. In addition, cell motility assay was used to determine the change of single cell motility upon BNIP3 knockdown. Similarily, hypoxia enhanced the motility of CFPAC-1 cells, while BNIP3 knockdown notably downregulated the cell motility under both normoxia and hypoxia condition ( Figure 1J and 1K). Moreover, results of the EdU staining and CCK-8 assay revealed that cell proliferation was increased when subjected to hypoxic microenvironment. The inhibition of BNIP3 expression decreased the proliferation of CFPAC-1 cells under both normoxia and hypoxia ( Figure 1L, 1M, 1N). These results together indicated that BNIP3 promoted the migration and proliferation of CFPAC-1 cells.

Autophagy was activated in pancreatic cancer cells under hypoxia
Previously, a great deal of literatures demonstrate that hypoxic microenvironment is an important factor that enhances cell autophagy in solid tumors. Next, we investigated the autophagy flux in CFPAC-1 cells under hypoxia. As shown in Figure 2A and 2B, hypoxia elevated expressions of LC3-II, an important autophagic marker, in a time-dependent manner. To determine whether hypoxia-induced LC3-II requires the transcriptional activity in CFPAC-1 cells, the cells were pretreated with an inhibitor of mRNA synthesis, actinomycin D (ACTD), for 1 h followed by hypoxic treatment for 48 h. ACTD notably suppressed the increase of hypoxia-induced LC3-II and p62 expression ( Figure 2C, 2D, 2E), indicating that initial increase in LC3-II and p62 levels depends on the activation of transcriptional machinery under hypoxia. Furthermore, the indirect immunofluorescence staining of endogenous LC3 showed that hypoxia treatment increased the autophagic puncta in CFPAC-1 cells ( Figure 2F). Consistently, in LC3-mRFP-GFP adenovirus transfected CFPAC-1 cells, more autolysosomes (red puncta) and autophagosomes (yellow puncta) were found in hypoxia group compared to normoxia group ( Figure 2G). Furthermore, to determine whether hypoxia induces autophagic flux, CFPAC-1 cells were treated with a lysosomal inhibitor, chloroquine (CQ), which primarily increases the lysosomal pH, decreases the activity of degradative enzymes and consequently causes the accumulation of vesicular organelles in the cytoplasm, thus inhibiting autophagy. CQ treatment apparently elevated the LC3-II levels in cells under hypoxic conditions compared to normoxic conditions ( Figure 2H, 2I, 2J), which indicated the increase in autophagic flux under hypoxia. These observations revealed that autophagy was activated in pancreatic cancer cells under hypoxia.

BNIP3 involved in the autophagic activation of pancreatic cancer cells under hypoxia
As reported previously, BNIP3 is proved an autophagy regulator in a variety of cancer cells. To explore whether the BNIP3 involves in the activation of autophagy in CFPAC-1 cells under hypoxia, we firstly investigated the interaction of BNIP3 and LC3. Results of immunofluorescence staining demonstrated that the co-localization (yellow dots) of BNIP3 (red dots) and LC3 (green dots) was obviously increased ( Figure 3A). Meanwhile, co-immunoprecipitation assay showed a direct interaction of BNIP3 and LC3 in hypoxic CFPAC-1 cells ( Figure 3B). Then Western blot was performed to detect the role of BNIP3 for autophagy in CFPAC-1 cells under hypoxia. As depicted in Figure 3C and 3D, BNIP3 knockdown significantly decreased the level of LC3-II, indicating the autophagy inhibition. BafA1 is a specific V-ATPase inhibitor that neutralizes lysosomal pH and block lysosomal degradation. Treating with BafA1 further elevated p62 and LC3-II in CFPAC-1 cells under hypoxia, while BNIP3 knockdown suppressed p62 and LC3-II expression by BafA1 ( Figure 3E, 3F, 3G).
Moreover, the indirect immunofluorescence staining of endogenous LC3 demonstrated that knockdown of BNIP3 expression inhibited the formation of hypoxia-induced autophagic puncta in CFPAC-1 cells ( Figure 3H). Consistently, in the CFPAC-1 cells transfected with LC3-mRFP-GFP adenovirus, less autophagosomes (yellow puncta) and autolysosomes (red puncta) were found in hypoxia þ siBNIP3 group compared to hypoxia þ siNC group ( Figure 3I). Furthermore, cells were transfected with Ad-GFP-LC3 and Ad-Dsred-Mito to evaluate the mitophagy. The colocalization of LC3 and Mitotracker decreased following BNIP3 knockdown under hypoxia but no change was found under normoxia, which suggested that BNIP3 promoted the mitophagosome formation under hypoxia in CFPAC-1 cells ( Figure S1). These results indicated that BNIP3 was responsible for the activation of autophagy and mitophagy in the CFPAC-1 cells under hypoxia.

Autophagy regulates BNIP3-induced pancreatic cancer cell migration and proliferation under hypoxia
As is well-known that Atg5 involves in the Atg12-Atg5 ubiquitin-like conjugation and direct the elongation and sealing of the autophagosome membrane, Atg7 is an E1-like ligase that conjugates Atg5 to Atg12 [19]. To explore whether autophagy involved in CFPAC-1 cell migration and proliferation under hypoxia treatment, siRNA targeting Atg5 (siAtg5) or Atg7 (siAtg7) was transfected into the cells to suppress autophagy specifically, as confirmed with Western blot ( Figure 4A, 4B, 4C, 4B; Figure S2A and S2B). Specific knockdown of Atg5 or Atg7 in CFPAC-1 cells significantly suppressed the LC3-II expression, which revealed the  inhibition of autophagy ( Figure 4C, 4D, 4E; Fig.S2C, S2D, S2E). Meanwhile, Atg5 knockdown obviously inhibited the LC3-II expression by BNIP3 overexpression ( Figure S4A). Basing on the above-mentioned results, we explored if autophagy regulated BNIP3-induced pancreatic cancer cell migration and proliferation under hypoxia. A scratch wound healing assay and a single cell motility assay were respectively performed to investigated cell migration and motility upon the autophagic impairment. Results of the scratch wound model showed that hypoxia significantly promoted the cell migration, while an obvious wound gap remained in the CFPAC-1 cells with Atg5 knockdown (Figure 4F and 4G). As shown in Figure 4H and 4I, knockdown of Atg5 remarkably decreased the range of cell trajectory and the velocity of cell movement, which was in line with the results obtained from the scratch wound healing assay. Moreover, results of EdU staining and CCK-8 assay revealed that the knockdown of Atg5 expression decreased the proliferation of CFPAC-1 cells under both normoxia and hypoxia ( Figure 1J, 1K, 1L). Similar results were found in CFPAC-1 cells with Atg7 knockdown (Figure S2F, S2G, S2H, S2I, S2J, S2K, S2L). Furthermore, Atg5-targeting siRNA was transfected in CFPAC-1 cells with BNIP3 overexpression to inhibit autophagy. As shown in Figure S4B and S4C, Atg5 knockdown resulted in a larger wound gap, a smaller range of cell trajectory and a lower speed of cell movement compared with the BNIP3-overexpressed group. Results of the EdU staining and CCK-8 assay demonstrated a significant reduction of cell proliferation upon Atg5 knockdown in BNIP3-overexpressed group ( Figure S4D and S4E). Together, these results suggust that autophagy involves in the BNIP3-induced pancreatic cancer cell migration and proliferation under hypoxia.

ERK1/2 signaling pathway involves in the regulation of BNIP3autophagy axis under hypoxia
To gain further insights into the mechanisms responsible for the activation of BNIP3-autophagy in the hypoxia-induced CFPAC-1 cell migration under hypoxia treatment, we first investigated the activation of ERK1/2 signaling pathway. CFPAC-1 cells were randomized into four groups and pre-subjected to hypoxia (1% O 2 ) for 12, 24 or 48 h before the detection of ERK1/2 activation with the Western blot assay. Notably, hypoxia enhanced the ERK1/2 activation in a time-dependent manner, and achieved the maximum effect by hypoxia for 24 h ( Figure 5A and 5B). Then a selective chemical inhibitor was used to suppress the activation of ERK1/2 signaling. The addition of PD98058 decreased the expressions of BNIP3 and LC3-II under normoxia condition ( Figure S3A and S3B), and it was significant that hypoxia-induced elevation of BNIP3 and LC3-II were both ameliorated by the inhibition of ERK1/2 signaling as shown by Western blot ( Figure 5C and 5D), which was also confirmed with the immunofluorescence staining under confocal microscope ( Figure 5E, 5F, 5G). Moreover, the overexpression of BNIP3 by lentivirus infection could reversed the downregulation of LC3-II by the inhibition of ERK1/2 pathway ( Figure S5A). These results suggested that ERK1/2 signaling pathway participate in the activation of BNIP3-autophagy axis in CFPAC-1 cells under hypoxia.
Next, the EdU staining and CCK-8 assay were used to evaluate the proliferation of indicated CFPAC-1 cells. As shown in Figure 5H and 5I and Figure S3C-S3E, inhibition of ERK1/2 induced a remarkable decrease in the proliferation of CFPAC-1 cells under normoxia and hypoxia. The single cell motility assay showed that inhibition of ERK1/2 significant attenuated the cell motility, as represented by the inhibition in the range of cell trajectory and the velocity of cell movement ( Figure 5J and 5K and Figure S3F and S3G). Consistently, results of the scratch wound healing assay demonstrated an obvious wound gap remained in the CFPAC-1 cells with ERK1/2 inhibition ( Figure 5L and 5M and Figure S3H and S3I). Furthermore, BNIP3 overexpression reversed the impairment of cell migration and proliferation by ERK1/2 inhibition ( Figure S5B, S5C, S5D, S5E). Taken with the results above, inhibition of ERK1/2 signaling attenuated the elevation of BNIP3 expression, as well as the activation of autophagy, and consequently suppressed the proliferation and migration of CFPAC-1 cells under hypoxia.

BNIP3 enhanced the migration and proliferation of Panc1 cells under hypoxia
In order to test whether BNIP3 generally promotes the migration and proliferation of pancreatic cancer cells, we test its role in Panc1 cells under hypoxia. The Panc1 cells were transfected with lentivirus or siRNA for the overexpression or knockdown of BNIP3. As expected, BNIP3 in Panc1 cells was significantly overexpressed by AdBNIP3 infection and downregulated with siBNIP3 transfection, respectively. The upregulation of BNIP3 induced the increase of LC3-II, while the knockdown of BNIP3 impaired the elevation of LC3-II under hypoxia ( Figure 6A), which suggested a similar decrease of autophagy under hypoxia as in the CFPAC-1 cells. Then, a wound healing assay ( Figure 6B) and a cell motility assay ( Figure 6C) were used to evaluate the effects of BNIP3 on the migration of Panc1 cells. Results demonstrated that BNIP3 overexpression promoted the cell migration, hypoxia promoted the migration and of Panc1 cells, while BNIP3 knockdown suppressed the cell migration under hypoxia. Moreover, results of EdU staining and CCK-8 assay revealed that BNIP3 overexpression increased the cell proliferation under normoxia, inhibition of BNIP3 expression decreased the proliferation of Panc1 cells under hypoxia ( Figure 6D, 6E, 6F). These results indicated that BNIP3 promoted the migration and proliferation of Panc1 cells.
In order to evaluate the involvement of autophagy in BNIP3-induced migration and proliferation of Panc1 cells, Atg5-siRNA was transfected in the BNIP3-overexpressed Panc1 cells. Western blot showed a significant downregulation of the BNIP3-induced LC3-II expression with the knockdown of Atg5 ( Figure 6G). Results of the wound healing assay ( Figure 6H and 6I) and cell motility assay ( Figure 6J) showed a significant inhibition of the BNIP3-induced migration. Next, the EdU staining and CCK-8 assay was applied to investigate the proliferation of indicated Panc1 cells. As shown in Figure 6K, 6L, 6M, the BNIP3-induced proliferation of Panc1 cells was attenuated by the knockdown of Atg5. These results indicated that autophagy acted a role in the BNIP3-induced migration and proliferation of Panc1 cells under hypoxia.
Then, the role of ERK1/2 pathway in BNIP3/autophagy-regulated migration and proliferation of Panc1 cells was investigated. As depicted in Figure 6N, inhibition of ERK1/2 pathway obviously suppressed the expressions of BNIP3 and LC3-II under hypoxia, while the overexpression of BNIP3 by lentivirus infection could reversed the downregulation of   (caption on next page) LC3-II by the inhibition of ERK1/2 pathway. Results of the wound healing assay ( Figure 6O) demonstrated an obvious wound gap remained in the Panc1 cells with ERK1/2 inhibition under hypoxia, BNIP3 overexpression significantly promoted the healing of the "scratched wound". Similarly, the single cell motility assay showed that inhibition of ERK1/2 significant decreased the range of cell trajectory and the velocity of cell movement ( Figure 6P). Furthermore, the EdU staining and CCK-8 assay were applied to investigate the cell proliferation. As shown in Figure 6Q, 6R, 6S, inhibition of ERK1/2 induced a remarkable decrease in the proliferation of Panc1 cells under hypoxia, which could be promoted by the BNIP3 overexpression. Taken together, these results revealed that the ERK1/2 signaling elevated the BNIP3 expression and activated autophagy, which consequently promoted the proliferation and migration of Panc1 cells under hypoxia.

Discussion
Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly cancers, while little advances in treatment are reported in the past decades. PDAC contains abundant stromal cells, lacks vascularization, thus persistent hypoxia is accompanied within the tumors [1]. It has been reported that hypoxic microenvironment induces the aggressive characteristics of cancer cells in many types of cancer, including pancreatic cancer, while the potential molecular mechanisms need further studies in-depth.
BNIP3 is a hypoxia-responsive protein and plays an important role in the progress and metastasis of multiple cancers. As a pro-apoptotic atypical BH3-only protein, BNIP3 involves in cell necrosis, apoptosis, or autophagy depending on the cellular context. Recently, BNIP3 has been demonstrated to promote keratinocyte migration in an autophagydependent way [20]. Consistently, the results here showed that the expressions of BNIP3 and the hallmarks of autophagy were remarkably increased in CFPAC-1 and Panc1 cells under hypoxia, accompanied with the elevations of cell migration and proliferation, as determined respectively with the scratched wound-healing assay, single-cell motility assay, EdU staining and CCK-8 assay. Knockdown of BNIP3 with siRNA transfection significantly impaired the proliferation and migration of CFPAC-1 cells, as well as the autophagy flux as determined with Western blot and immunofluoresent staining. Moreover, the interaction of BNIP3 and LC3-II was revealed with CO-IP and co-localization assay.
Furthermore, the hypoxia-induced CFPAC-1 migration and proliferation was notably suppressed by the inhibition of autophagy with the Atg5-or Atg7-targeted siRNAs. Meanwhile, the overexpression of BNIP3 by the lentivirus infection enhanced the migration and proliferation of CFPAC-1 and Panc1 cells, which could be disturbed by Atg5 knockdown. These results together indicated that BNIP3-mediated autophagy promoted the migration and proliferation of pancreatic cancer cells.
BNIP3 expression is usually transcriptionally regulated by hypoxiainducible factor 1α (HIF-1α), a major transcription factor in hypoxia [7]. To date, studies have revealed several HIF-1αÀindependent mechanisms that mediate BNIP3 expression. For example, BNIP3 expression under energy stress and in pathological cardiac hypertrophy is modulated by forkhead box (FOX)O transcription factor [21,22]; Sp3 might be the major transcriptional regulator of BNIP3 in prostate cancer [23]. The mitogen-activated protein kinase (MAPK) cascades transduce the signaling of stress-induced stimuli or extracellular growth factor, and play crucial roles for the control of cell migration, proliferation, and survival [24,25]. In hepatocellular carcinoma, phosphorylation of JNK closely binds to the promoter of BNIP3 gene and contributes to BNIP3 expression [26]. As recently reported, BNIP3 expression was upregulated by P38 and JNK signaling in keratinocytes under hypoxia [20]. In this study, we reported that extracellular-signal-regulated kinase (ERK) played a role in the upregulation of BNIP3 expression in hypoxic pancreatic cancer cells. Maybe hypoxia-induced ERK phosphorylation can also bind to the promoter of BNIP3 gene and regulate its expression, which remains further investigations.
Autophagy is a conserved process in eukaryotic cells that recycles the altered or unused organelles and cellular components and delivers them to the lysosome for degradation. Autophagy has been reported to involve in migration and growth of both normal and cancerous cells, while the molecular mechanisms remains largely unknown. Previously, autophagy has been shown to regulate recycling of β1 integrin membranes, depolymerization of focal adhesions, and acquisition of mesenchymal markers, which are essential for cell migration [27,28,29,30,31]. Mitochondria are potential targets for hypoxia-induced autophagy. The mitochondria dynamics provide energy supplement for the reorganization of cytoskeleton and sustaining the cell migration [32]. Autophagy has been shown to be upregulated in PDAC and involved in resistance to cytotoxic chemotherapy and targeted therapy, while the molecular mechanisms that activate autophagy in PDAC have only just begun to Graph quantifying the rate of wound closure. The results were presented as the means AE SEM. *P < 0.05 vs. Hypo þ AdNC group, # P < 0.05 vs. Hypo þ AdNC þ ERKi group. (P) Single cell motility assays were performed to detect cell motility of indicated groups. Representative images of cell trajectories were shown (n ¼ 3). Graph quantifying the average velocity of cell movement. Results were shown as means AE SEM. *P < 0.05 vs. Hypo þ AdNC group, # P < 0.05 vs. Hypo þ AdNC þ ERKi group. (Q) Cell proliferation was evaluated with EdU staining (green) (n ¼ 3). Nuclei were stained with DAPI (blue). (R) Graph quantifying the number of EdU-positive cells in (Q). (S) CCK-8 assay was further performed to test the cell proliferation and the results were presented as the means AE SEM. *P < 0.05 vs. Hypo þ AdNC group, # P < 0.05 vs. Hypo þ AdNC þ ERKi group. All the experiments were repeated 3 times. Norm, normoxia; Hypo, hypoxia. emerge. Recently, Wong et al. showed that phosphatase PP2A increased the activity of UNC-51-like kinase 1 (ULK1) and participated in the initiation of autophagy, which was also responsible for increased autophagy in PDAC cells [33]. In addition, Settembre et al. reported that the starvation-induced TFEB nuclear translocation in HeLa cells was suppressed by ERK/MAPK2-mediated phosphorylation [34], which was also observed in PDAC models that inhibition of RAS-RAF-MEK-ERK/MAPK signaling pathway resulted in increased autophagic flux [35,36]. These results indicate that ERK/MAPK signaling is clearly involved in autophagy regulation of PDAC. Here, our results demonstrated a BNIP3-dependent way of autophagy activation in CFPAC-1 and Panc1 cells under hypoxia. The ERK/MAPK signaling pathway was obviously upregulated under hypoxia, and the elevations of BNIP3 expression and activation of autophagy was both suppressed by the ERK/MAPK inhibitor, which consequently inhibited the cell migration and proliferation. Moreover, BNIP3 overexpression significantly reversed the impairment of cell migration and proliferation, as well as the activation of autophagy, by ERK1/2 inhibition. The above observations indicate that autophagy is a therapeutic target for PDAC, and the combination of ERK/MAPK pathway inhibitors and autophagy inhibitors may be an effective treatment for PDAC.
Together, our current work demonstrated that BNIP3-induced autophagy was involved in the migration and proliferation of PDAC cells under hypoxia. Meanwhile, ERK/MAPK signaling is significant activated and enhances BNIP3-mediated autophagy in CFPAC-1 cells under hypoxia, and consequently promotes PDAC cell migration and proliferation. These results reveal a new molecular explanation for the autophagy activation in PDAC.

Author contribution statement
Xiaojiao Wang: Conceived and designed the experiments; Contributed reagents, materials, analysis tools or data; Analyzed and interpreted the data; Wrote the paper.
Hongmei Li; Can Zhang: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper. Qiong Zhang; Jiezhi Jia: Performed the experiments; Analyzed and interpreted the data.

Funding statement
Xiaojiao Wang was supported by National Natural Science Foundation of China [82103441].

Data availability statement
The data that has been used is confidential.

Declaration of interest's statement
The authors declare no conflict of interest.

Additional information
Supplementary content related to this article has been published online at https://doi.org/10.1016/j.heliyon.2022.e11190.