The serine/threonine kinase STK33 has been implicated in cancer cell proliferation. Here, we provide evidence of a critical role for STK33 in the pathogenesis and metastatic progression of pancreatic ductal adenocarcinoma (PDAC). STK33 expression in PDAC was regulated by the hypoxia-inducible transcription factor HIF1α. In human PDAC specimens, STK33 was overexpressed and associated with poor prognosis. Enforced STK33 expression promoted PDAC proliferation, migration, invasion, and tumor growth, whereas STK33 depletion exerted opposing effects. Mechanistic investigations showed that HIF1α regulated STK33 via direct binding to a hypoxia response element in its promoter. In showing that dysregulated HIF1α/STK33 signaling promotes PDAC growth and progression, our results suggest STK33 as a candidate therapeutic target to improve PDAC treatment. Cancer Res; 77(24); 6851–62. ©2017 AACR.
Pancreatic ductal adenocarcinoma (PDAC) is the seventh leading cause of cancer-related deaths worldwide, with incidence rates matching mortality rates (1, 2). Its poor prognosis is reflected by a 5-year survival rate around 7%–8% despite significant improvement of treatment and development of new treatment modalities, such as chemotherapies and immunotherapies (1, 3–6). Pancreatic cancer progression involves a complex regulatory network consisting of many signaling pathways. Previous studies uncovered multiple mechanisms underlying PDAC pathogenesis (7, 8). However, the molecular mechanisms that trigger PDAC growth and progression remained largely unknown.
The tumor microenvironment has a key role in the initiation and progression of PDAC, and hypoxia is a prominent physicochemical feature of tumor microenvironments (9–11). PDAC is hypoxic, primarily owing to its hypovascular nature, driving its drug resistance and high malignancy (12–14). Hypoxia activates signaling pathways to balance out the abnormal tumor vasculature and increases oxygen consumption associated with dysregulated tumor cell proliferation. At the molecular level, this adaptation relies primarily on the transcription factor hypoxia-inducible factor (HIF)-1α. It is a master regulator of cellular and systemic homeostatic response to hypoxia, which transcriptionally regulates many genes involved in energy metabolism, angiogenesis, and apoptosis as well as genes involved in oxygen delivery or metabolic adaptation to hypoxia (15–18). Although previous studies verified multiple HIF1α target genes, novel and potentially important candidate targets of HIF1α in tumor development and progression are continually being identified.
Serine/threonine kinase 33 (STK33) belongs to a family of calcium/calmodulin–dependent kinases. STK33 is expressed in a variety of normal tissues, including the testis, fetal lung and heart, and retina; however, it is weakly expressed in the normal pancreas (19). Researchers have observed elevated expression of STK33 in a variety of cancers (20–28). Over the past decade, many studies focused on the “synthetic lethality” function of STK33 in tumors with KRas mutations, suggesting that STK33 is a druggable target for Kras-mutant cancer patients, when therapeutic targeting of KRas remains one of the biggest challenges in cancer research (20, 22). Other studies refuted this conclusion, though, challenging the necessity of STK33 expression in mutant KRas-driven cancer development (27). Recent biofunctional analysis of STK33 demonstrated that it plays an active role in promoting tumor initiation, progression, malignancy, and resistance to therapy. These studies using BRD-8899, an effective kinase inhibitor of STK33, identified kinase-dependent and -independent regulatory effects of STK33 on a variety of malignant tumors (23, 26). However, the expression of and potential roles for STK33 in PDAC development and progression have yet to be examined.
In this study, we found that STK33 was highly expressed in PDACs and significantly correlated with poor prognosis. Hypoxia induced STK33 expression in PDAC cells, and STK33 was a direct target of and transcriptionally regulated by HIF1α. Therefore, this novel HIF1α/STK33 signaling pathway critically regulates PDAC growth and metastasis and is a novel PDAC biomarker for use in new treatment modalities.
Materials and Methods
Human tissue specimens and IHC analysis
The expression of STK33 and HIF1α was analyzed using tissue microarrays (TMA) containing 100 primary PDAC and 78 normal adjacent pancreatic tissue specimens (SuperChip). The characteristics of the TMAs were listed in Supplementary Tables S1 and S2. IHC analyses of these specimens were conducted with anti-STK33 (catalog no. ab206296) and anti-HIF1α (catalog no. ab113642) antibodies (Abcam) as described previously (29). Overall survival (OS) was defined as the interval from the date of surgery to the date of death. All analyses for human subjects were approved by the institutional review board of The University of Texas MD Anderson Cancer Center (Houston, TX) and conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all subjects wherever necessary.
Cell culture and hypoxic conditions
The HPNE, 293T, and human PDAC cell lines AsPC-1, BxPC-3, Capan-2, PANC-1, and SW1990 were purchased from the ATCC. The human PDAC cell line FG and murine pancreatic cancer cell lines Panc02 and Panc02-H7 were described previously (30). All the cell lines were obtained between 2012 and 2015 and authenticated by ATCC using short tandem repeat profiling analysis, routinely tested for mycoplasma contamination within the last 6 months by using Hoechst staining and PCR, and used at passage numbers <10 for this study after reception or thawing in our laboratory. All of these cell lines were maintained in plastic flasks as adherent monolayers in Eagle Minimal Essential Medium supplemented with 10% FBS, sodium pyruvate, nonessential amino acids, l-glutamine, and a vitamin solution (Flow Laboratories). All cells were incubated at 37°C in a humidified atmosphere with 5% CO2. For hypoxic culture, cells were placed in a hypoxia incubator in an atmosphere consisting of 94% N2, 5% CO2, and 1% O2.
Plasmids and siRNAs
The plasmids pcDNA3.0-STK33 (pSTK33) and pcDNA3.0-hemagglutinin (HA)-HIF1α were purchased from OriGene and Addgene. siRNAs for STK33 (siSTK33) and HIF1α (siHIF1α) were synthesized by Invitrogen. A negative control siRNA (Invitrogen) and control pcDNA3.0 vector were used. Transfection of plasmids and siRNAs into PDAC cells was performed using Lipofectamine 2000 CD transfection reagent (Invitrogen). For transient transfection, cells were transfected with plasmids or siRNA at different concentrations as indicated for 48 hours before performance of functional assays.
Western blot analysis
Standard Western blotting was carried out using whole-cell protein lysates; primary antibodies against STK33 (catalog no. #4F7; Abnova), HIF1α (catalog no. ab113642; Abcam), and hemagglutinin (catalog no. 26183-1MG; Thermo Fisher Scientific); and a secondary antibody (anti-rabbit IgG or anti-mouse IgG; Santa Cruz Biotechnology). Equal protein sample loading was monitored using an anti-actin antibody.
Male and female pathogen-free athymic nude mice and C57BL/6 mice were purchased from the National Cancer Institute (Bethesda, MD). The animals were maintained in facilities approved by AAALAC International in accordance with the current regulations and standards of the U.S. Department of Agriculture and Department of Health and Human Services. All animal studies have been conducted in accordance with the Institutional Animal Care and Use Committee of The University of Texas MD Anderson Cancer Center.
Tumor growth and metastasis
Human PDAC cells (1 × 106) in 0.1 mL of Hank balanced salt solution were injected subcutaneously into the right scapular regions of nude mice. The resulting tumors were measured every week. Tumor-bearing mice were killed by cervical dislocation when they became moribund or on day 35 after injection, and their tumors were removed and weighed. Panc02 and Panc02-H7 cells were subcutaneously injected into syngeneic C57BL/6 mice (1 × 105 cells per mouse). Mice were killed 21 (Panc02) or 18 (Panc02-H7) days after tumor injections, and their tumors were removed and weighed.
To quantitatively measure metastasis, an experimental liver metastasis model was used. Specifically, tumor cells (5 × 105) were injected intravenously into another group of mice via the ileocolic vein. All mice were sacrificed by cervical dislocation on day 28 after injection or when they appeared to be moribund. Their livers were then removed, and surface metastases on the livers were counted after dissecting them into their individual lobes. Every surface metastasis was examined by two investigators who were unaware of the experimental protocol and scored separately.
Tumor cell migration/invasion assay
Both cell scratch–wound (horizontal migration) assays and modified Boyden chamber (vertical invasion) were performed to determine the migratory ability and invasiveness of PDAC cells with altered STK33 expression as described previously (31). For the cell scratch–wound assays, cells were grown in 6-well plates until confluent. A wound was generated on the surface of the resulting cell monolayer via scraping with the 10-μL tip of a pipette. After that, the cells were incubated for 12–48 hours. The cells in the wounded monolayer were photographed at different time points, and cell migration was assessed by measuring gap sizes in multiple fields. For the Boyden chamber assay, 24-well tissue culture plates with 12-cell culture inserts (Millipore) were used. Each insert contained an 8-μm pore-size polycarbonate membrane with a precoated thin layer of a basement membrane matrix (ECMatrix for the invasion assay) or without a coated matrix (for the migration assay). Ten percent FBS-containing medium was placed in the bottom chambers to act as a chemoattractant. Cells (5 × 105) in a 300-μL volume of serum-free medium were placed in the top chambers and incubated at 37°C for 48 hours. Cells on the bottom surface of the polycarbonate membrane, which had invaded the ECMatrix and migrated through the membrane, were stained, counted, and photographed under a microscope.
Reverse transcription-PCR analysis of STK33 mRNA expression was performed using total RNA. Total RNA was purified using an RNeasy Plus Mini Kit (catalog no. 74134; Qiagen), and cDNA was synthesized using an iScript cDNA Synthesis Kit (catalog no. 170-8890; Bio-Rad). The cDNA product was subjected to PCR amplification with PCR primers to detect each factor. PCR primers used in this report were summarized in Supplementary Table S3.
Construction of STK33 promoter reporter plasmids
A 719-bp DNA fragment containing the STK33 5′ sequence from -722 to -4 relative to the transcription initiation site was subcloned into a pGL4.16-basic vector (Promega). The resulting full-length reporter plasmid, which contained multiple hypoxia response elements (HRE), was designated pGL4.16-634 (the promoter reporter plasmid was named by the start site of the HRE). Deletion mutation reporters for this plasmid (pGL4.16-436, and pGL4.16-347) were then generated. All of these constructs were verified by sequencing the inserts and flanking regions of the plasmids.
Promoter reporter and dual luciferase assay
PDAC cells were transfected with STK33 promoter reporters, siRNAs, or specific gene expression plasmids. The STK33 promoter activity in these cells was normalized via cotransfection of a β-actin/Renilla luciferase reporter containing a full-length Renilla luciferase gene. The resulting luciferase activity in the cells was quantified using a dual luciferase assay system (Promega) 24 hours after transfection.
Chromatin immunoprecipitation assay
PDAC cells (2 × 106) were prepared for a chromatin immunoprecipitation (ChIP) assay using a ChIP assay kit (Millipore) according to the manufacturer's protocol. The resulting precipitated DNA specimens were analyzed using PCR to amplify fractions of the STK33 promoter. The PCR products were resolved electrophoretically on a 2% agarose gel and visualized using ethidium bromide staining.
A two-tailed χ2 test or the Fisher exact test was used to determine the significance of differences among covariates. All in vitro experiments were performed in triplicate and at least three times. Data were presented either as means ± SD from one representative independent experiment of three with similar results or means ± SEM from three independent experiments. The significance of the in vitro and in vivo data was determined using the Student t test (two-tailed) or one-way ANOVA. In all of the tests, P values less than 0.05 were considered statistically significant. The SPSS software program (version 17.0; IBM Corporation) was used for statistical analysis.
Direct association of elevated expression of STK33 with pathologic features of PDACs
To determine the roles of STK33 in PDAC pathogenesis, we first investigated STK33 protein expression in the 100 primary PDAC specimens, 78 tumor-adjacent pancreatic tissue specimens, and normal pancreatic tissue specimens in the TMAs. The clinicopathologic characteristics of the TMAs are listed in Supplementary Table S1. As shown in Fig. 1A and B, elevated STK33 expression was associated with poor histologic differentiation (total, P < 0.001). We observed that the STK33 protein expression in PDAC specimens was much higher than that in normal tissue specimens (Supplementary Fig. S1). Western blot analysis of the expression of STK33 in paired normal pancreatic tissue and PDAC specimens confirmed this finding (Fig. 1C). Furthermore, we found that patients with high STK33 expression had shorter OS than did those with low expression (P < 0.001; Fig. 1D). We also investigated the relationship of STK33 protein expression with age, sex, tumor size, T category, and regional lymph node metastasis and found no statistically significant differences in expression according to these variables (P > 0.05; Supplementary Table S2). These findings indicated that STK33 plays critical roles in PDAC development and progression and may be a valuable biomarker for this disease.
The subcellular localization of STK33 in PDACs
We examined STK33 expression and its subcellular location in PDAC and tumor-adjacent tissue specimens using IHC, observing that STK33 was located in both the cytoplasm and nucleus (Supplementary Fig. S1). Also, we confirmed the localization of STK33 in PDAC cells using immunofluorescent staining and found that STK33 was mainly localized in the cytoplasm in AsPC-1 and Capan-2 cells but in the nuclei in FG, BxPC-3, PANC-1, and SW1990 (Supplementary Fig. S2). Interestingly, both PDAC cell lines with cytoplasmic (AsPC-1) and nuclear (PANC-1) STK33 expression exhibited a remarkable upregulation of nuclear STK33, but only slight change of cytoplasmic STK33, which may indicated a special role of nuclear expression of STK33 in PDAC progression (please see Supplementary Fig. S3A). To determine the clinical significance of cytoplasmic and nuclear expression of STK33, we further analyzed its relationships with clinical parameters. Results showed that patients with nuclear expression of STK33 had significantly shorter OS survival durations (P = 0.000; Fig. 1E), whereas the expression levels of cytoplasmic STK33 expression were not significantly correlated with OS of PDAC patients (Supplementary Fig. S3B).
Promotion of PDAC growth by STK33 expression in vitro and in vivo
To determine the effect of STK33 expression on PDAC growth, we induced its expression in PDAC cell lines. STK33 was highly expressed in FG, PANC-1, and SW1990 cells but expressed at relatively low levels in AsPC-1, BxPC-3, and Capan-2 cells (Fig. 2A). As shown in Fig. 2B, pSTK33 effectively induced overexpression of STK33 in AsPC-1 and Capan-2 cells, and siSTK33 markedly downregulated expression of STK33 in SW1990 and PANC-1 cells. Consistent results were obtained when AsPC-1 and Capan-2 were transfected with siSTK33 and PANC-1 and SW1990 were transfected with pSTK33 (Supplementary Fig. S4). We then investigated the effect of STK33 on PDAC biology using pSTK33 and siSTK33#2. We found that overexpression of STK33 in AsPC-1 and Capan-2 cells led to increased monolayer growth, whereas knockdown of expression of STK33 resulted in decreased cell monolayer growth (Fig. 2C).
In animal models, transfection with pSTK33 consistently promoted the growth of AsPC-1 cells in the subcutis, and knockdown of STK33 expression inhibited the growth of SW1990 cells (Fig. 2D–F). The impacts of altered expression of STK33 on tumor growth in xenograft model were further confirmed in syngeneic mouse model of mouse pancreatic cancer (Fig. 2G). These data demonstrated that STK33 promoted the growth of PDACs in vitro and in vivo, supporting that STK33 may function as an oncogene in PDAC cases.
Promotion of PDAC invasion and migration by STK33 in vitro and in vivo
To determine the effect of STK33 expression on PDAC migration and invasion, we transfected AsPC-1 and Capan-2 cells with pSTK33 and PANC-1 and SW1990 cells with siSTK33#2 for 48 hours. We had wounded the transfected cells via scratching, and maintained them for at least 12 hours. The results demonstrated that forced expression of STK33 strongly promoted the flattening and spreading of AsPC-1 (Fig. 3A) and Capan-2 (Fig. 3B) cells, whereas downregulation of expression of STK33 attenuated the flattening and spreading of SW1990 (Fig. 3C) and PANC-1 (Fig. 3D) cells. Boyden chamber assay results confirmed these findings. Both the migratory ability and invasiveness of STK33-transfected AsPC-1 (Fig. 3A) and Capan-2 (Fig. 3B) cells were much greater than those of control cells, whereas those of siSTK33-transfected SW1990 (Fig. 3C) and PANC-1 (Fig. 3D) cells were markedly attenuated. We then confirmed the in vitro results in mouse models of PDAC, finding that enforced expression of STK33 markedly increased liver metastasis of AsPC-1 cells (Supplementary Fig. S5A), whereas knockdown of expression of STK33 abrogated liver metastasis of SW1990 cells (Supplementary Fig. S5B). Similarly, enforced expression of STK33 markedly increased lung metastasis of AsPC-1 cells (Supplementary Fig. S6A), whereas knockdown of expression of STK33 abrogated lung metastasis of SW1990 cells (Supplementary Fig. S6B). These data further confirmed the oncogenic role of STK33 in progression and metastasis of PDAC.
STK33 expression is upregulated under hypoxic conditions in PDAC cells
To determine whether STK33 expression can be induced by hypoxia, we exposed AsPC-1, Capan-2, and PANC-1 cells to hypoxic conditions (1% O2) for up to 48 hours. We found that the STK33 expression of PDAC cells at the mRNA and protein levels increased markedly under these conditions and HIF1α protein expression increased rapidly in these cell lines (Fig. 4A and B). We also examined the expression of HIF1α and STK33 in PDAC cells after treatment with CoCl2, a known HIF1α activator. We found that the treatment increased the levels of HIF1α and STK33 expression in a dose-dependent manner in AsPC-1 and Capan-2 cells (Fig. 4C and D). Furthermore, Matrigel invasion assay demonstrated that knockdown of STK33 expression partially inhibited the invasion of AsPC-1 and Capan-2 cells under hypoxic conditions (Fig. 4E). These results suggested that hypoxia induces STK33 expression in PDAC cells and HIF1α is involved in this regulatory mechanism.
Close relationship between HIF1α and STK33 expression in PDAC cells
To identify the mechanisms underlying STK33 overexpression in PDAC cells, we investigated the effect of altered expression of HIF1α, the best characterized regulator in hypoxic microenvironments, on expression of STK33 in PDAC cells. As shown in Fig. 5A, overexpression of HIF1α in AsPC-1 and Capan-2 cells markedly upregulated the expression of both STK33 mRNA and protein, whereas knockdown of expression of HIF1α decreased them in SW1990 and PANC-1 cells. Subsequently, we performed IHC analysis of HIF1α expression using a TMA with specimens obtained from the same PDAC patient cohort used for STK33 staining. On the basis of immunostaining for HIF1α and STK33, we observed a close correlation between the protein expression levels for STK33 and HIF1α (Fig. 5B). Importantly, statistical analysis revealed that the expression of STK33 was positively correlated with that of HIF1α in the PDAC specimens (R = 0.68; P = 0.000; Fig. 5C). Double fluorescence staining assay revealed a colocalization of STK33 and HIF1α in PDAC tissues (Supplementary Fig. S7). Moreover, similar to the results on the impact of STK33 expression on patient survivals (Supplementary Fig. S8A), elevated expression of HIF1α in this study was correlated with a reduced OS durations (P = 0.001 and P = 0.000, according to two different grouping methods; Supplementary Fig. S8B and S8C). It was also demonstrated that the combination of STK33 and HIF1α expression increased our ability to predict poor prognosis for PDAC (P = 0.0097; Supplementary Fig. S8D).
To understand the role of STK33 in HIF1α–mediated PDAC cell migration, we overexpressed STK33 in HIF1α–knockdown SW1990 and PANC-1 cells. Results showed that STK33 overexpression at least partially rescued the inhibitory effect of HIF1α knockdown on SW1990 and PANC-1 cell migration, suggesting that STK33 was involved in HIF1α–mediated migration (Fig. 5D).
Transcriptional regulation of STK33 expression by HIF1α in PDAC cells
To further explore the mechanism of regulation of STK33 expression by HIF1α, we analyzed the STK33 promoter sequence 5′-CGTG-3′ for potential HREs, which were described previously (32). Sequence analysis of the STK33 promoter uncovered three putative HREs located at -347 (HRE1), -436 (HRE2), and -634 (HRE3) bp relative to the transcriptional start site of STK33 (Fig. 6A). We then generated the full-length STK33 promoter pSTK33-634 and deletion mutants of it. To determine whether HIF1α regulates STK33 expression at the transcriptional level, we cotransfected the deletion mutant reporters with or without HIF1α expression vectors into 293T cells. Luciferase reporter assay results demonstrated that deletion of the region from -520 to -399 bp, covering the HRE2 site, markedly reduced the promoter activity of STK33 induced by HIF1α (Fig. 6B). To further determine whether HIF1α regulates STK33 promoter transcriptional activity in PDACs, we cotransfected a pGL4.16-634 reporter with HIF1α expression vectors or siRNA into PDAC cells. As shown in Fig. 6C, increased HIF1α expression in AsPC-1 and Capan-2 cells activated STK33 promoter activity, whereas knockdown of HIF1α expression in SW1990 and PANC-1 cells attenuated the STK33 promoter activity. To provide more evidence that HIF1α bound directly to the STK33 promoter, we conducted a ChIP assay using chromatin prepared from AsPC-1 and SW1990 cells. The results confirmed that HIF1α directly bound to the HRE2 site (Fig. 6D) rather than other HRE sites in the STK33 promoter in PDAC cells. These data strongly suggested that HIF1α bound to the STK33 promoter and transcriptionally regulated STK33 expression in PDAC cells.
In this study, we investigated the biologic effects of and underlying molecular basis for STK33 expression in PDACs and delineated the clinical significance of the newly identified HIF1α/STK33 regulatory pathway. We found that expression of STK33 was frequently higher in tumors than in nontumorous adjacent tissue. We also demonstrated a positive correlation between STK33 expression and clinicopathologic features of PDAC patients, particularly that increased STK33 expression predicts poor tumor differentiation and decreased survival OS durations. In vitro and in vivo experiments demonstrated that STK33 had a potent oncogenic role in promoting PDAC proliferation, migration, and invasion. Our mechanistic studies revealed that STK33 overexpression in PDACs was mainly regulated by HIF1α, which binds directly to HREs on the STK33 promoter. Given these results, we concluded that STK33 is a direct target of HIF1α in PDACs, constituting a new signaling axis and promoting PDAC development and progression.
Recent studies have demonstrated elevated expression of STK33 in a variety of cancers, including hepatocellular carcinoma, large-cell lung cancer, and hypopharyngeal squamous cell carcinoma. However, the expression and biologic function of STK33 in PDACs during tumorigenesis has been seldom studied. We found that STK33 expression was markedly increased in PDAC cell lines and specimens. Analysis of the relationship of clinicopathologic characteristics of the PDAC patients from whom the TMA specimens were obtained with STK33 expression demonstrated that the expression was associated with tumor differentiation and poor prognosis. Previous studies demonstrated that STK33 is localized mainly in the cytoplasm of cells in different organs (19). Our IHC results demonstrated that STK33 localized to the nuclei and cytoplasm of the PDAC cells. Interestingly, nuclear expression of STK33 appeared to be associated with reduced OS durations. Further studies are required to determine the mechanism of STK33 protein subcellular localization and translocation of STK33 to the cytoplasm and nucleus. These results indicated that STK33 may function as an oncogene and play important roles in PDAC development and progression. In our biologic studies, we found that elevated expression of STK33 markedly enhanced PDAC proliferation, migration, and invasion in vitro and tumor growth in vivo, whereas silencing of STK33 markedly decreased the ability of PDAC cells to proliferate, migrate, and invade and inhibited the growth of PDACs in vivo. All of these findings were in accordance with those of previous studies, strongly supporting that STK33 functions as an oncogene in PDACs.
KRas is frequently mutated in many types of cancer cells (33). Clinical observations and functional studies suggested that KRas is an attractive therapeutic target for PDAC treatment. However, effective KRas inhibitors have yet to be found (34–36). Researchers identified STK33 as having a synthetic lethality function in the context of in cells expressing mutant KRas using high-throughput RNA interference (21). Also, authors reported that knockdown of STK33 expression induced apoptosis in KRas-dependent cancer cell lines but not in cells wild-type for KRas (21, 22). Because therapeutic targeting of R has been a great challenge to date, this finding led authors to propose that inhibitors of STK33 kinase activity would selectively kill Kras-mutant cells in cancer patients. However, over the past decade, this conclusion was opposed by many researchers, as they obtained no likely results in their laboratories, so the effect of anti-KRas agents that inhibit STK33 remains controversial (23, 26, 37). Answering this question is significant to PDAC, because the vast majority of pancreatic cancers harbor KRas mutations (∼90%). In this study, we determined the biologic function of STK33 in KRas-mutant cell lines (AsPC-1, Capan-2, PANC-1, and SW1990). However, a deficiency of evidence from the use of wild-type PDAC cell models in our study leaves the validity of correlation of STK33 and Ras expression in PDACs uncertain.
Researchers discovered STK33 in the course of sequencing the human chromosome 11 region 11p15 and mouse chromosome 7 (38). Ample studies have proven that STK33 has potent oncogenicity and preliminarily identified its protumor mechanism. As a kinase, SKT33 exhibited autophosphorylation and directly phosphorylated the intermediate filament protein vimentin, which functions as a structural component of and modulates signal transduction in cancer cells (20, 39). Authors also reported that the kinase-independent effect of STK33 supports STK33 as a critical regulator of cancer cell proliferation and migration (23, 26). Using a transgenic mouse model of hepatocellular carcinoma, Yang and colleagues (26) found that STK33 bound directly to the transcriptional factor C-myc and increased its transcriptional activity, which could not be inhibited by treatment with the STK33 kinase inhibitor BRD-8899. However, the C-terminus of STK33 played an inhibitory role in regulating this interaction and significantly inhibited hepatocellular carcinoma proliferation in vitro and tumor growth in vivo. In addition, Azoitei and colleagues (22) found that the HSP90/CDC37 chaperon complex bound to and stabilized STK33 in cancer cells by preventing its degradation from by the proteasome-mediated pathway. However, the exploration of potential mechanisms of STK33 in regulating PDAC biology has been limited.
Increasing evidence indicates that hypoxia is an important driving force for PDAC progression (7, 40–44). HIF1α, a subunit of HIF-1, functions as a master regulator of cellular and systemic homeostatic response to hypoxia (45–48). HIF1α has long been known to be a transcriptional factor, inducing the expression of multiple target genes in favor of the tolerance of PDAC cells to hypoxia, by recognizing HREs (5′-RCGTG-3′). Investigators found that in a sustained hypoxic environment, HIF1α was stabilized and prevented from undergoing protein degradation in an ubiquitin/proteasome pathway (49). In this study, we found that in PDAC cells, the expression of STK33 increased markedly under hypoxic conditions (1% O2) and that expression of HIF1α was upregulated. We confirmed these results in our treatment of PDAC cells with CoCl2 at different concentrations. Furthermore, PDAC cells exhibited enhanced migration under hypoxic but not normoxic conditions, and this enhancement was attenuated considerably by knockdown of STK33 expression. These results suggested that HIF1α is involved in upregulation of STK33 expression in PDAC cells.
We found four lines of evidence in this study verifying the presence of the HIF1α/STK33 signaling pathway in PDACs and supporting its role in regulation of PDAC progression and metastasis. First, we found that HIF1α and STK33 were concomitantly overexpressed in pancreatic tumor specimens. Second, overexpression of HIF1α upregulated the expression of STK33 at both the mRNA and protein level, whereas reduced expression of HIF1α did the opposite. Third, HIF1α bound directly to the STK33 promoter region and regulated expression of STK33 at the transcriptional level. Fourth, overexpression of STK33 rescued inhibition of PDAC migration by HIF1α knockdown. All of these results strongly supported that STK33 expression in PDACs was transcriptionally regulated by HIF1α.
In summary, we obtained both clinical and experimental evidence identifying STK33 as an important oncogene in PDACs and found that its expression was frequently increased in PDAC cell lines and specimens. Biologically, STK33 expression promoted PDAC proliferation, migration, and invasion in vitro and tumor growth in vivo. Mechanistically, the expression of STK33 in PDACs was transcriptionally activated by HIF1α, an important regulator of hypoxic microenvironments. Thus, this study is fundamentally important, as we not only identified a novel molecular mechanism of PDAC growth and progression but also identified the aberrant HIF1α/STK33 signaling pathway as a promising new molecular target for novel therapeutic modalities that inhibit PDAC metastasis and progression. The mechanism of nuclear translocation of STK33 and its potential relationship with KRas mutations in PDAC cells warrant further exploration.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Conception and design: F. Kong, X. Deng, D. Xie, Z. Li, K. Xie
Development of methodology: Y. Chen, X. Deng, L. Li, D. Xie
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): F. Kong, X. Kong, J. Zhu, J. Du, L. Li, Z. Li
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): F. Kong, X. Kong, Y. Du, J. Du, K. Xie
Writing, review, and/or revision of the manuscript: F. Kong, X. Kong, Y. Du, K. Xie
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): F. Kong, J. Du, Z. Jia, Z. Li, K. Xie
Study supervision: Z. Li, K. Xie
We thank Don Norwood for editorial assistance.
This work was supported by grants R01CA172233, R01CA195651, and R01CA198090 from the National Cancer Institute, NIH (to K. Xie) and grant NSFC no. 81402017 and Shanghai Yang Fan Project # 14YF1405600 (to X. Kong) and grant NSFC no. 81402425 (to L. Li) from the National Natural Science Foundation of China.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.