Background
The protein kinase D (PKD) family of serine/threonine kinases belongs to the Ca
2+/calmodulin-dependent protein kinase (CaMK) superfamily. The three isoforms (PKD1, 2, 3) of PKD are widely distributed in a variety of tissues and show high sequence homology [
1‐
3]. Several conserved structure domains are present in PKD, including a diacylglycerol-binding C1 domain and a PH domain that exerts an autoinhibitory function to the kinase activity. PKD can be activated by PKC-mediated trans-phosphorylation of two conserved serine residues (Serine 738/742 in human PKD1) in the activation loop of PKD [
4]. Sustained PKD activation can be maintained via PKC-independent autophosphorylation events [
5]. Through the DAG/PKC/PKD axis, PKD plays an important role in propagating signals from G protein-coupled receptors (GPCRs) and growth factor receptors at the cell surface.
PKD has been implicated in multiple cancers. Altered PKD expression and activity have been demonstrated in prostate, breast, pancreas, skin, and gastric cancers [
1,
6]. PKD1 is the most intensely studied PKD isoform to date. In certain cancers including pancreatic and skin cancer, higher PKD1 expression and activity were detected in tumors as compared to normal tissues and increased PKD1 expression was associated with hyper-proliferative phenotype and increased tumor aggressiveness [
6,
7]. Interestingly, in other cancer types including breast, gastric and prostate cancer, PKD1 was found to be downregulated in primary tumors or metastases [
8‐
11]. Reduced PKD1 expression was associated with increased tumor invasiveness and its overexpression in prostate cancer cells was shown to inhibit tumor cell proliferation [
10,
12]. Thus, the functional relevance of PKD1 to tumor initiation and progression remains to be determined.
Head and neck squamous cell carcinoma (HNSCC) is one of the most common type of human cancers. The annual incidence is more than 500,000 cases worldwide. There are more than 40,000 new cases of HNSCC reported in the United States, and nearly 12,000 will die from the disease [
13]. The origin of HNSCC involves multiple organs, including the oral cavity, pharynx, and larynx. HNSCC at early-stage (Stage I and II) can be curatively treated with surgery or radiotherapy. However, advanced HNSCC (stage III and IV) remains an aggressive disease that is associated with high morbidity and mortality. The 3-year survival rate for patients with advanced disease under standard therapy is only 30–50%, and a large number of these patients (nearly 40% to 60%) subsequently develop locoregional recurrences or distant metastases [
13‐
15]. Despite the advances in treatment strategies, the survival rates for patients with advanced HNSCC have not improved significantly, underscoring an urgent need to better understand the molecular mechanisms underlying the pathogenesis of HNSCC. The role of PKD in HNSCC has not been fully investigated. The current study was undertaken to evaluate the expression of PKD1 in HNSCC tumor specimens and cell lines to gain insights into its clinical significance. The study also sought to investigate the functional implication of PKD1 in HNSCC by systematically determining its cancer-associated biological properties in HNSCC cells in vitro and in vivo and to assess the potential value of targeting PKD1 for cancer therapy.
Methods
Materials
Doxycylcline hyclate, 5-Aza-2′-deoxycytidine (5-aza-dC), gastrin-releasing peptide, and DMSO were obtained from Sigma-Aldrich (St. Louis, MO). The histone deacetylase HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) was purchased from Cayman Chemical (Ann Arbor, MI). Bombesin was obtained from Fisher Scientific (Pittsburgh, PA).
Immunohistochemistry (IHC)
The normal and malignant human head and neck tissue sections were obtained from US Biomax (Rockville, MD,
www.biomax.us) and Pantomics (Richmond, CA,
www.pantomics.com). The patient-paired tumor specimens were obtained from Dr. Yehai Liu at the Department of Otolaryngology, Head and Neck Surgery, First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China. A written informed consent from the donor or the next of kin was obtained for the use of these samples in research. The tissues were collected following a named medically prescribed procedure during standard treatment without authors’ involvement. The fully randomized and de-identified samples were provided to the authors. These samples are exempt from the requirement of IRB approval (Exempt Category 4) since they are de-identified and publicly available. For IHC staining, the tissue sections were dewaxed with xylene and rehydrated through gradient ethanol into water. For antigen retrieval, sections were heated in citrate buffer (pH 6.0) for 10 min at 95 °C. The sections were then digested with 0.05% trypsin for 10 min at 37 °C. Endogenous peroxidase activity was quenched with 0.3% H
2O
2 in methanol for 30 min at room temperature. After washing with PBS, slides were pre-blocked with 10% normal goat non-immune serum at 37 °C for 30 min. Sections were incubated with primary antibody targeting PKD1 (1:150) at 4 °C overnight, washed with PBS, and incubated with biotinylated secondary antibody at a 1:200 dilution for 30 min. The sections were then developed by incubating first in Vectastain ABC reagent (Vector Laboratories, Inc., Burlington, CA) and then with 3,3′-diaminobenzidine (Sigma-Aldrich). Slides were counterstained with hematoxylin, dehydrated, and mounted on coverslips. Negative controls were obtained by omitting the primary antibody. The staining was scored independently by two experienced researchers according to the number and intensity of immunopositive cells in a blinded fashion. The percentage of positive tumor cells was determined semi-quantitatively by assessing the entire tumor section and each sample was scored based on the following criteria: 0 (0–4%), 1 (5–24%), 2 (25–49%), 3 (50–74%), or 4 (75–100%). The intensity of immunostaining was categorized as 0 (negative), 1+ (weak), 2+ (moderate) or 3+ (strong). A final immunoreactive score between 0 and 12 was calculated by multiplying the two scores. These procedures were adapted from previously published studies [
16‐
18].
Cell lines and culture conditions
HNSCC cell lines UMSCC-1, UMSCC-10A, UMSCC22B, Cal33, UPCI 4B, UPCI 15B, 1483 and 686LN were obtained from Dr. Jennifer R. grandis (University of California, San Francisco, CA) as described previously [
19]. Het-1A (CRL-2692), a human esophageal squamous epithelial cell line, was obtained from ATCC (Manassas, VA) and cultured in Airway Epithelial Cell Basal Growth Medium with supplement mix (Promo Cell, Heidelberg, Germany). UMSCC-1, Cal33, UMSCC-10A, UMSCC22B, UPCI 4B, UPCI 15B and 1483 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (Fisher Scientific, Pittsburgh, PA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA), penicillin (100 U/ml)/streptomycin (100 μg/ml) at 37 °C in a humidified atmosphere of 5% CO
2. OSC19 cells were cultured in Eagle’s Minimum Essential Medium (EMEM) (Fisher) plus 10% FBS and Non-Essential Amino Acid (Fisher). 686LN cells were maintained in Ham’s F-12 medium (Fisher) containing 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin. All cell lines were authenticated by the Research Animal Diagnostic Laboratory by species-specific PCR testing within 6 months of use.
Development of stable doxycycline (Dox)-inducible PKD1 expression cell lines
The doxycycline-inducible PKD1 expression cell lines were developed using the Tet-On 3G System from Clontech (Mountain View, CA). Briefly, wild-type or constitutive-active (CA) PKD1 gene was sub-cloned into a pTRE3G-based expression vector to generate the pTRE3G-PKD1 or pTRE3G-PKD1-CA plasmid. Meanwhile, UMSCC-1 and 686LN cells were transfected with the pCMV-Tet3G plasmid and selected with G418 to generate stable Tet-On 3G cell lines that constitutively expressed the Tet-On 3G transactivator protein. The stable Tet-On 3G cell lines were then transfected with the pTRE3G-PKD1 or pTRE3G-PKD1-CA plasmid, along with a linear selection marker for puromycin. The cells were then selected with puromycin to generate double-stable cell lines that expressed PKD1 or PKD1-CA in response to Dox treatment. The stable clones were isolated and the induction of PKD1 was confirmed by Western blotting analysis. Optimal Dox induction condition was determined in a time- and concentration-response experiment, and 500 and 50 ng/ml for 48 h were selected as the optimal induction conditions for UMSCC-1 and 686LN cells, respectively.
Western blotting
Western blotting analysis was conducted as previously described [
20]. Primary antibodies used for Western blotting were from the following sources: PKD1, p-S916-PKD1, p-S744/748-PKD1 antibodies were obtained from Cell Signaling Technology (Danvers, MA); p-Ser742-PKD1 antibody was from Invitrogen (Carlsbad, CA); PKD2 antibody was from Abcam (Cambridge, MA); glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was obtained from Enzo (Farmingdale, NY); tubulin antibody was from Santa Cruz Biotechnology (Santa Cruz, CA).
Quantitative real-time RT-PCR
Total RNA was prepared using RNeasy Mini Kit according to manufacturer’s instructions (Qiagen, Valencia, CA). Reverse-transcription and real-time quantitative PCR were performed as previously described [
21]. Sequences of the primer pairs used were as follows: PKD1, CGCACATCATCTGCTGAACT (forward) and CTTTCGGTGCACAACGTTTA (reverse); PKD2, GGGCAGTTTGGAGTG GTCTA (forward) and ACCAGGATCTGGGTGATGAG (reverse); PKD3, CATGTCCACCAGGAACCAAG (forward) and GACGGGTGTAAGAGTGAACAGC (reverse); GAPDH, GCAAATTCCATGGCACCGT (forward) and TCGCCCCACTTGATTTTGG (reverse). The PCR protocol included 1 cycle at 95 °C for 30 s, then 40 cycles of a 95 °C for 5 s step followed by 5 s at either 62 °C or 65 °C, depending on the optimal annealing temperature for each primer set. Melt curves were conducted to assure specificity of the primer sets as well as absence of primer-dimers.
Transient knockdown of PKD1 was achieved using multiple siRNAs targeting different regions of PRKD1 gene. Two validated Stealth PKD1 siRNAs (si-PKD1–1 and si-PKD1–2) and a BLOCK-iT PKD1 siRNA (si-PKD1–3: GUCGAGAGAAGAGGUCAAATT) were obtained from Invitrogen. The sequence for the PKD2-targeting siRNA (si-D2–2) is UCAUCACCCAGAUCCUGGUGGCUUU. The HNSCC cell lines were transiently transfected using DharmaFECT Reagent 3 (Dharmacon, Lafayette, CO) according to the manufacturer’s instructions. Cells were harvested after two days and the levels of PKD1 or PKD2 knockdown were assessed by Western blotting analysis.
Cell proliferation assay, wound healing assay, and Matrigel invasion assay
Cell proliferation was determined for UMSCC-1 cells transfected with PKD1 siRNAs and stable PKD1-inducible UMSCC-1 and 686LN clones by counting cell numbers for seven consecutive days as previously reported [
22]. Growth media with or without Dox was refreshed every 2 days. Cell migration was measured by wound healing assay as previously described [
23]. The average % wound healing was determined based on 4 measurements of the wound area. Cell invasion was determined by Matrigel invasion assay as described before [
24]. For stable inducible clones, cells were incubated with Dox for 48 h prior to seeding in BD Matrigel invasion or control inserts (BD Biosciences, San Jose, CA). Dox was added to the top and bottom chambers of control and invasion inserts.
Subcutaneous xenograft mouse model
Six-week old female athymic (NCr) nu/nu mice (NCI-Frederick Cancer Research Facility, Frederick, MD) were randomized into two groups (10 mice/group) for injection of control cells expressing empty vector (control group) or cells expressing stable inducible PKD1 (PKD1 group). The cells (4 × 10
6 cells) mixed 1:1 with Matrigel (BD Biosciences) were injected subcutaneously into both flanks of mice. Once tumors were palpable, mice in each group were divided to receive either drinking water or Dox-containing driving water (1 mg/mL). Water was changed every 2 days. Tumor size and mouse weight were monitored 2–3 times per week. Tumor size was measured as described [
21]. The experiment was terminated after 25 days and tumors were dissected for subsequent analysis. All animal studies were conducted in accordance with IACUC guidelines at the University of Pittsburgh.
Statistical analysis
All statistical analyses were performed using GraphPad Prism software. The significance between data points from cell proliferation, wound healing, and invasion experiments was assessed by Student’s t-test. The Mann-Whitney-Wilcoxon test was used for the tumor xenograft study. A p-value of < 0.05 was considered statistically significant.
Discussion
The PKD family has been implicated in a variety of biological processes associated with cancer initiation and progression. PKD1, the most extensively studied PKD isoform, has been shown to be dysregulated in a number of cancer types and plays important roles in tumor cell biology [
1,
6,
24,
25]. Interestingly, both tumor suppressive and tumor promoting functions of PKD1 have been reported, which couples to its up- or down-regulation in these tumors, implying a tumor type-specific function of PKD1 in cancer. Specifically, it has been shown that PKD1 is downregulated in invasive human breast tumors as compared to normal breast tissues. Overexpression of constitutively-active PKD1 inhibits the invasion of breast tumor cells, while knockdown of PKD1 confers invasiveness to non-invasive breast cancer cells, an effect that is potentially mediated through negative regulation of MMP expression [
8,
40]. In gastric cancer, PKD1 expression is decreased in gastric tumors and cell lines due to PKD1 promoter hypermethylation, and knockdown of PKD1 increased the invasiveness of gastric tumor cell lines [
11]. In prostate cancer, PKD1 was downregulated in androgen-independent prostate cancer and increased PKD1 expression blocks cell proliferation and motility [
9,
10,
12]. In human osteosarcoma, PKD1 expression in osteosarcoma is significantly lower than that in benign schwannoma samples, and the expression pattern correlated with metastatic potential [
41]. Overexpression of PKD1 inhibits osteosarcoma cell proliferation, invasion, and migration and reduced matrix metalloproteinase 2 (MMP2), while knockdown of PKD1 has the opposite effects. Overexpression of PKD1 has also been shown to suppress the growth of osteosarcoma xenografts in vivo [
41]. On the other hand, several studies in pancreatic cancer and basal cell carcinoma have demonstrated an opposite role of PKD1 in cancer. PKD1 has been shown to be upregulated both in expression and activity in pancreatic ductal adenocarcinoma as compared to normal pancreatic ducts [
27,
42]. The activation of PKD1 promotes pancreatic cancer cell proliferation and increased PKD1 expression contributes to therapy resistance [
6,
27,
43‐
45]. Meanwhile, overexpression of PKD1 significantly promoted DNA synthesis, anchorage-dependent/−independent growth, tumor cell invasion, and angiogenesis in pancreatic cancer cells [
44,
45]. In skin cancer, increased PKD1 expression has been demonstrated in basal cell carcinoma lesions as compared to normal epidermis [
7]. PKD1 has been associated with pro-proliferative and anti-differentiative phenotypes in epidermis and keratinocytes, implying that PKD promotes hyperproliferative disorders of the skin [
7,
46,
47].
HNSCC originates from the mucosal lining of the head and neck regions and accounts for 90% of head and neck cancers. There have not been any studies investigating the role of PKD in head and neck cancer. In this study, we conducted systematically analysis on the expression and function of PKD1 in HNSCC. Our data revealed that the expression of PKD1 was significantly lower in localized HNSCC tumors and metastases, a finding that was further confirmed in patient-paired tumor tissues where PKD1 was downregulated at both mRNA and protein levels in tumors as compared to the normal mucosa. Interestingly, reduced PKD1 expression was re-expressed in only one of five HNSCC cell lines following treatment with histone deacetylase and/or DNA methyltransferase inhibitors. This finding was consistent with the results obtained from cBioPortal analysis of 530 HNSCC tumors from TCGA where low level of DNA methylation on PRKD1 gene was indicated. Additionally, based on the TCGA data, genetic alteration (LOH or homozygous deletion) only accounts for a fraction of PKD1 mRNA downregulation (~ 13%). Thus, the mechanisms responsible for the downregulation of PKD1 mRNA and protein expression in majority of HNSCC tumors remain to be determined.
Functional analyses of PKD1 using RNAi and stable inducible cell lines revealed that altered PKD1 expression did not significantly affect the proliferation, survival, migration, or invasion of HNSCC cells in the basal state. To ensure that the lack of function was not due to insufficient kinase activity associated with the wild-type protein, a constitutive-active PKD1 mutant was generated and introduced into HNSCC cells and similar results were obtained. Depletion of endogenous PKD1 also did not affect proliferation of UMSCC-1 cells. However, in spite of these findings, some discrepancies were noted when proliferation assays were conducted at different initial seeding density, for example, when the cells were seeded at lower density, such as in Fig.
7c (3000 cells/well), Dox-induced PKD1 appeared to reduce the proliferation of 686LN cells at basal level [comparing Dox-induced and un-induced cells in the absence of bombesin]. However, when the cells were seeded at higher density, such as in Fig.
4h (20,000 cells/well), no difference was observed among cells with or without PKD1 overexpression. Thus, there might be transient and context-dependent growth inhibition by PKD1 at certain cellular context, but this effect is not sustained. Overall, our data consistently showed that either knockdown or overexpression of PKD1 did not significantly alter the proliferation of HNSCC cells in vitro. However, interestingly, induction of PKD1 in vivo by Dox provided a slight growth advantage to the HNSCC tumor xenografts and resulted in a significant increase in final tumor weight in Dox-induced vs the non-induced tumors. This correlated to increased ERK1/2 and NF-κB signaling activity, and enhanced tumor cell proliferation in vivo. Later, we demonstrated that in the presence of a mitogen (bombesin or GRP) that activates PKD, overexpression of PKD1 potentiated the mitogenic effects of bombesin in HNSCC, and depletion of endogenous PKD2, the predominant PKD isoform expressed in HNSCC cells, abolished such effect. At molecular level, overexpression of PKD1 promoted bombein- or GRP-induced ERK1/2 activation, while knockdown of PKD2 reduced EKR1/2 activation. It has been shown that the mitogenic effects of GRP is mediated by the activation of the MEK/EKR1/2 MAPK pathway through transactivating EGFR in HNSCC cells [
34]. Our findings imply that PKD1 and PKD2 may contribute to the mitogenic effect of GRP and bombesin by facilitating the activation of ERK1/2. This is a novel interesting finding that unlike other reports showing a significant functional role that associates with altered PKD1 expression in different tumors, our data indicate that PKD1 has limited functional impact in the proliferation, survival, migration, and invasion of HNSCC cells at the basal state, despite frequent downregulation of
PRKD1 transcript and protein expression in HNSCC tumors and cell lines. Importantly, under in vivo condition or in the presence of an activating mitogen such as GRP or bombesin, PKD1 and PKD2 act to promote tumor cell proliferation. Our results and others suggest that the biological functions of PKD may be cancer type- and cell context- dependent. Perhaps the function of PKD is contingent on another yet unknown protein/pathway in certain cancer types. Meanwhile, high expression of another PKD isoform, such as PKD2, may substitute the need for other PKD isoforms. In a recent report in gastric cancer where low PKD1 and high PKD2 expression were detected in poorly differentiated adenocarcinoma, overexpression of PKD1 had no/minimal effects on tumor cell survival and proliferation, which is consistent with our findings [
26]. In HNSCC, PKD2 was the predominant PKD isoform expressed in HNSCC cells. PKD2 mRNA was upregulated in seven out of ten tumors vs normal in patient-paired HNSCC tissue specimens. Thus, it is possible that PKD2 plays a predominant role in the growth, survival, and motility of HNSCC cells, and these functions have compensated the loss of PKD1 in tumors, our data from PKD2-knockdown cells support this claim.
Acknowledgments
The authors would like to thank Dr. Adhiraj Roy for his technical assistance during the revision of this manuscript and Dr. Shiyuan Cheng for critical reading and helpful discussion of our paper.