Background
Trop2 is a cell surface glycoprotein belonging to the
TACSTD gene family and highly overexpressed by a variety of epithelial carcinomas with low to restricted expression in normal tissues [
1‐
6]. Clinical data has shown a positive correlation between Trop2 expression levels and tumor aggressiveness and metastasis, and a negative correlation with overall patient survival [
1‐
6]. Trop2 is highly conserved among species with a 79% identical amino acid composition between human and murine Trop2. This protein was initially found to be highly expressed in trophoblast cells, which arise from epithelial trophectoderm cells and become invasive, phagocytosing and displacing uterine epithelial cells. This allows for the penetration of the uterine stroma in order to establish vascular interactions with the maternal blood supply [
7,
8]. Trop2 expression has also been observed in murine and human prostate basal cells with stem cell characteristics [
9]. Basal stem progenitor cells with high Trop2 expression were shown to give rise to basal, luminal and even neuroendocrine cells
in vivo. A similar behavior has also been reported in hepatic oval cells which are considered facultative hepatic stem cells and shown to express Trop2 [
10]. It thus appears that Trop2 provides crucial signals for cells with a requirement for proliferation, survival and invasion such as trophoblast cells or cells with progenitor-like characteristics. These same characteristics might be conferred to cancer cells by overexpression of this surface glycoprotein.
Trop2 has recently been identified as an oncogene leading to the invasiveness and tumorigenesis of colon cancer cells, but the underlying signaling mechanisms activated by this protein are still unknown [
11]. It has been shown that cross-linking this protein with antibodies results in a significant rise in intracellular calcium [Ca
2+] from internal stores which could have a significant effect on the activation and progression of the cell cycle as well as activation of other signaling pathways [
12‐
15]. The cytoplasmic tail of Trop2 appears to play an important role in signaling. One study has shown the presence of a phosphatidylinositol 4,5-bis phosphate (PIP
2)-binding sequence highly homologous to that of gelsolin [
16]. Within this sequence there is a conserved serine residue which is phosphorylated by protein kinase C (PKC) [
17]. Thus, PKC and mitogen-activated protein kinases (MAPKs) including ERK1/2 may be involved in Trop2 induced tumor cell growth [
17,
18].
The purpose of this study was to determine the effects of murine Trop2 expression (mTrop2) in cancer cells and to start delineating the pathways activated by this molecule. We found that mTrop2 expression resulted in increased cell proliferation at low serum concentrations with an increased percentage of cells entering S phase. Expression of mTrop2 also led to increased cell migration, foci formation and anchorage independent growth and translated to increased tumor growth in both subcutaneous and orthotopic tumor models. mTrop2 expression also led to increased liver metastasis as well as increased levels of phosphorylated p42/p44
MAPK (ERK1/ERK2) which is a master regulator of the G
1- to S-phase transition [
18,
19]. This translated to a rise in cyclin D1 and cyclin E protein levels with a downregulation of p27. This study provides new evidence that Trop2 contributes to tumor pathogenesis at least in part by activating the ERK1/2 MAPK pathway which has important implications for a variety of cellular pathways as it can affect cancer cell proliferation, migration, invasion and survival [
18,
20‐
22].
Discussion
In the current study, we used murine Trop2 to investigate the effects of its expression on murine pancreatic cancer cell proliferation and tumor growth. We showed that mTrop2 expression in the murine pancreatic cancer line (Panc02) led to an increased number of cells entering S phase which resulted in increased cell growth at low serum concentrations. Similarly, there was an enhanced ability of cells expressing mTrop2 to migrate even without the presence of serum in the media. This low requirement for serum might be indicative that Trop2 transduces a survival signal in a growth-factor independent manner. Trop2 expression also led to foci formation in NIH3T3 cells showing that expression of this protein can lead to a loss of contact inhibition. Further evidence for the role of mTrop2 in cell proliferation and/or survival was observed in the improved ability of Panc02 cells to form colonies in soft agar. Panc02 cells normally form colonies in soft agar, but expression of mTrop2 increased the rate of colony formation and by day 3 there were already on average 25 colonies compared to 1 colony for the vector control group and these colonies did not arise from cell clumping. Such in vitro characteristics were further maintained in subcutaneous and orthotopic tumor models where Panc02-mTrop2 cells led to a significant increase in tumor growth and metastatic rate. It is therefore evident that mTrop2 increases the growth, aggressiveness and possibly survival signals inside the cell.
By using an AP-1 SEAP reporter assay as well as cell lysates from control and mTrop2 expressing cells, we were able to delineate an initial signaling pathway activated by mTrop2. mTrop2 expressing cells showed an increase in the levels of phosphorylated ERK1/2 suggesting an activation of this MAPK pathway. Cell division is a complex process involving an intricate network of regulatory pathways [
18]. One of these regulatory pathways is the ERK1/2 mitogen-activated protein kinase pathway which transduces extracellular signals into intracellular responses and is necessary for G
1- to S-phase transition. This MAPK pathway can be activated by a variety of stimuli including mitogens, cytokines, and growth factors which induce a transient rise in intracellular calcium [Ca
2+] from both internal and external stores. The cross-linking of Trop2 has previously been shown by others to result in a significant rise in cytoplasmic calcium [Ca
2+] and this could in turn be activating the MAPK pathway through activation of PKC and/or Ca
2+/calmodulin-dependent protein kinase II (CaMKII), both of which can modulate the ERK pathway [
12,
27,
28]. These two proteins are activated by an increase in Ca
2+ and CaMKII can bind and phosphorylate MEK1 leading to the activation of ERK [
27,
29]. The link between Trop2-induced calcium increase and activation of the ERK1/2 MAPK pathway has yet to be established.
It is important to note that downstream activation of AP-1 can be mediated not only by ERK activation, but also by JNK or p38 MAPKs [
30]. In this study we only focused on ERK activation due to the observed changes on cell growth and cell cycle progression observed following mTrop2 expression as well as the preferential involvement of ERK in the AP-1 SEAP assays. However, it is possible that crosstalk with the other MAPK pathways is taking place upstream of AP-1 as this transcription factor serves as a connecting node, linking various signal transduction pathways [
31]. Trop2 could therefore be affecting other MAPK pathways to some degree. Nonetheless, ERK signaling can activate AP-1 which can play an important role in cell proliferation, apoptosis, differentiation, cancer cell invasion and has been shown to regulate cyclin D1 and E2F in breast cancer cells [
31].
Upon phosphorylation of the activation loop residues of p44/p42 by MEK2, there is subsequent activation of downstream targets which include transcription factors and genes important for the cell cycle such as
cyclin D and
cyclin E [
32]. In the current study, an increase in cyclin D1 and cyclin E expression was indeed observed in Panc02 cells expressing mTrop2. Cyclin D1 partners with CDK4 and CDK6 in the early to mid-G
1 phase to phosphorylate and inactivate the retinoblastoma protein (pRB). The inactivation of pRB is also mediated by the cooperation of cyclin E/CDK2 both of which showed increased expression in mTrop2 expressing cells. Cyclin D1 and cyclin E are both key regulators of the G
1- to S-phase transition and have been implicated with tumorigenesis and metastasis [
33,
34]. The cyclin-dependent kinase inhibitor 1B, also known as p27, which binds to and prevents the activation of cyclin D1-CDK4 or cyclin E-CDK2 complexes, was also downregulated in mTrop2 expressing cells corroborating a progression of the cell cycle [
35].
Apart from a role in cell-cycle progression cyclin D1 could also be providing additional signals independent of CDK4/6 which are also implicated in tumorigenesis such as interaction with both FOXO1 and FOXO3a to inhibit anoikis [
33]. This inhibition could allow cells not only to survive and proliferate, but also to metastasize in the absence of an extracellular matrix support, something that was observed in our anchorage-independent growth assay and orthotopic murine model where Panc02-mTrop2 cells showed an improved capacity for anchorage-independent growth and an increased metastatic potential [
36]. Heightened ERK activity could also induce the phosphorylation of FOXO3a at residues S294, S344 and S425 promoting its cytoplasmic localization and proteasomal degradation following ubiquitination by MDM2 [
37]. This interaction between the ERK pathway and FOXO3a has been shown to promote cell growth and tumorigenesis, but whether Trop2 induced activation of ERK results in FOXO3a degradation still needs to be determined [
38]. Activation of ERK1/2 could also be providing anti-apoptotic signals thus promoting the survival of tumor cells [
20,
39].
The majority of the experiments presented here focused on the use of the murine pancreatic cancer cell line Panc02 and expression of the murine homolog of Trop2. Even though Trop2 is highly conserved among species and similarities between murine and human Trop2 suggest a conservation of protein structure and a conservation of intracellular signaling, there is a possibility that murine and human Trop2 might induce different effects in murine and human cancer cells respectively. It is therefore important to confirm the results presented here in multiple human pancreatic cancer cell lines expressing human Trop2.
It is evident that Trop2 expression increases the level of phosphorylated ERK1/2 which has downstream effects on various cellular functions. Inhibition of this pathway could have a significant effect on tumor cell growth. Targeting this MAPK pathway with the use of chemical inhibitors could potentially be used as a way to counteract at least some of the oncogenic effects mediated by this cell-surface glycoprotein and potentially affect Trop2 expressing tumor cells at metastatic sites. Inhibitors of the ERK pathway have already entered clinical trials as potential therapeutic agents, but ERK inhibitors can block a number of signals upstream of ERK [
40,
41]. In the case of pancreatic cancer, more than 90% of pancreatic adenocarcinomas show mutations in the
KRAS gene which result in constitutively active Ras, which can affect the activation of the ERK MAPK pathway [
42‐
44]. Therefore targeting ERK in pancreatic cancer patients will not specifically block signals from Trop2, but would rather block a number of signals which result in the activation of ERK such as those induced by
KRAS mutations. The use of ERK inhibitors in pancreatic cancer patients could therefore have no specific association with Trop2 and a specific inhibitor targeting Trop2 mediated signals would be highly desirable and could potentially augment the effects of ERK MAPK pathway inhibitors like PD0325901 and AZD6244 on pancreatic cancer cells. Further investigation into the signaling mechanisms and protein interactions mediated by Trop2 could lead to a better understanding of the important role this protein plays in cancerous cells. Precise protein interactions with its cytoplasmic tail as well as interactions with its extracellular region and studies aimed at determining the ligand for Trop2 could aid in the development of compounds specifically targeting Trop2 functions. The association of this molecule with prostate and hepatic oval cells displaying stem-cell characteristics hints to the possibility that Trop2 could potentially be present and used as a marker for cancer stem cells as has recently been reported for human prostate cancer [
45]. Whether Trop2 plays a role in deregulating characteristic stem cell proliferation and differentiation pathways such as Notch, hedgehog and Wnt deserves further attention. If Trop2 is indeed expressed by cancer stem cells, targeting and thoroughly understanding the mechanistic pathways affected by this molecule becomes of further importance.
Methods
Cell culture and antibodies
Panc02 murine pancreatic adenocarcinoma cells were originally established by Corbett et al. by implanting cotton threads into the pancreas of C57BL/6 mice which were impregnated with 3-methylcholanthrene [
46]. These cells were a kind gift from Dr. Sabry el-Naggar (Medical University of South Carolina) and were maintained in DMEM supplemented with 5% fetal bovine serum (FBS) (HyClone), 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco). NIH3T3 and 4T1 cells were a kind gift from Dr. Paul Ling and Dr. Adrian Lee (Baylor College of Medicine) and were maintained in DMEM supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin (complete DMEM). MC38 murine colorectal adenocarcinoma cells were a kind gift from Dr. John C. Morris (National Cancer Institute). These cells were maintained in RPMI 1640 medium supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were grown at 37°C in 5% CO
2. The human colonic epithelial cell line HCT-116 was obtained from ATCC (CCL-247) and maintained in complete DMEM media. Human pancreatic ductal epithelial cells (HPDE) previously described by Furukawa et al. were maintained in keratinocyte serum-free (KSF) medium supplemented with bovine pituitary extract and epidermal growth factor (Gibco) [
47].
The following antibodies and dilutions were used: anti-p44/42 MAPK (Thr202/Tyr204) 1:1000 (Cell Signaling, 9101), anti-cyclin D1 1:500 (Santa Cruz, sc-20044), anti-p27 1:1000 (Cell Signaling, 2552), anti-CDK2 1:1000 (Cell Signaling, 2546), anti-CDK4 1:1000 (Cell Signaling, 2906), anti-cyclin E 1:500 (Upstate Cell Signaling Solutions, 07-687), goat anti-rabbit IgG, HRP-linked 1:2000 (Cell Signaling, 7074) and goat anti-mouse IgG, HRP-linked 1:2000 (Cell Signaling, 7076).
Stable cell lines
To generate stable Panc02 cells expressing mTrop2, full-length mTrop2 cDNA (BC117720) was cloned into the lentiviral vector pWPXLd (Addgene). Lentivirus harboring the mTrop2 gene was generated by cotransfecting the 2nd generation packaging vector psPAX2 (Addgene), the envelope containing plasmid pMD2.G (Addgene) and pWPXLd-mTrop2 into 293FT cells. For control lentivirus normal pWPXLd was utilized. Viral supernatants were collected, filtered, concentrated and used to infect Panc02 cells. Cells were selected based on their expression of mTrop2 or eGFP as measured by real-time RT-PCR, immunoblotting and flow cytometry. This procedure was used for the other murine cell lines as well (4T1 and MC38).
For the generation of stable HCT-116 and HPDE cells overexpressing human Trop2 (hTrop2) a pBabe-hTrop2 vector was utilized. This vector was a kind gift from Dr. Loren Michel (Washington University School of Medicine). Retrovirus harboring either the pBabe or pBabe-hTrop2 constructs were generated and used for the infection of cells followed by selection with puromycin.
Immunohistochemistry
For immunohistochemical staining tumor and liver tissue samples were extracted and fixed overnight in formalin. The next day samples were washed in 70% ethanol and embedded in paraffin. Sections (5 μm) were then cut and mounted onto glass slides followed by overnight incubation at 55°C. The tissues were then deparaffinized and rehydrated with xylene and graded alcohol series. Antigen retrieval was performed by using 10 mM sodium citrate buffer (pH 6) for 20 min. Endogenous peroxidases were quenched by incubating slides for 20 min in methanol containing 30% hydrogen peroxide. Samples were then blocked for 1 hr followed by overnight incubation of primary antibodies at 4°C. The antibody dilutions used were: anti-murine Trop2 1:40 (R&D Systems, AF1122), anti-Ki-67 1:1000 (Novacastra, United Kingdom), anti-PCNA 1:500 (Santa Cruz, sc-7907), anti-cyclin D1 1:500 (Santa Cruz, sc-20044) and anti-cyclin E 1:500 (Upstate Cell Signaling Solutions, 07-687). Slides were then washed in PBS followed by incubation with biotinylated secondary antibodies for 30 min. Stain was visualized by incubating slides for 30 min with ABC reagent followed by diaminobenzidine (DAB) treatment for 2-5 min (Vector Laboratories).
SEAP reporter assay
Partially confluent 293T cells (70-80%) were co-transfected with 200 ng of AP-1 secreted alkaline phosphatase (SEAP) reporter gene plasmid DNA (pAP-1 SEAP), 500 ng of expression vector DNA (pWPXLd, pWPXLd-mTrop2) or positive control vector (pSH-1 SEAP) with Fugene HD transfection reagent (Roche) in 24-well plates. After 24 hours media was removed and serum-free media added to each well. The next day media was collected and assayed for SEAP activity using a FLUOstar Optima fluorescence plate reader (BMG Labtech).
Proliferation assays
For the proliferation assay, 2000 cells/well were seeded in flat-bottom 96-well plates in complete DMEM containing 5% FBS. The next day, cells were serum-starved for 24 h followed by the addition of 0.2% FBS. Cells were cultured for 3 or 5 days, at which point 20 μl of 3-(4,5-dimethyl-thiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) (Promega, Madison, WI) was added to each well and incubated at 37°C for 1.5 h. Absorbance was recorded at 490 nm with an EL-800 universal microplate reader (Bio-Tek Instruments, Winooski, VT).
For the proliferation assay in the presence of the MEK1 inhibitor PD98059 (Calbiochem, San Diego, CA), serum starvation was released by the addition of DMEM containing 0.2% FBS and PD98059 (1 μM) for 4 h. After incubation cells were carefully washed twice and kept in DMEM with 0.2% FBS. Cells were cultured for 3 days followed by MTS analysis.
Cell cycle analysis
Cells were serum-starved for 24 h followed by the addition of media containing 2% serum and collected after 4 or 8 h. Cells were harvested and processed using the CycleTEST PLUS DNA reagent kit (Becton Dickinson, Franklin Lakes, NJ) following the manufacturer's instructions. Briefly, cells were washed three times with buffer containing sodium citrate, DMSO and sucrose. Cells were subsequently incubated for 10 min each in solution A (enzymatic digestion of cell membranes and cytoskeletons), solution B (inhibit trypsin activity and digest RNA) and solution C (stoichiometric binding of propidium iodide to DNA at a final concentration of 125 ug/ml). Cells were analyzed by flow cytometry using a FACSCalibur (Becton Dickinson) and FlowJo ver. 7.2.1 (Tree Star, Ashland, OR).
Wound healing assay
Wounds were generated in confluent cell monolayers grown in 6-well plates with media containing either 0% or 5% FBS using a sterile pipette tip. Healing was observed at 0, 24, and 48 h along the scrape line and a representative field for each cell line was photographed.
NIH3T3 cells were plated at 5 × 105 cells/well in a 6-well plate. Cells were transfected with 1 μg of pWPXLd or pWPXLd-mTrop2 using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). NIH-3T3 cells expressing mTrop2 or GFP were then seeded in triplicate at 1 × 105 cells/well in a 6-well plate. Cells were allowed to grow and fed three times a week until foci with a diameter larger than 1 mm appeared. Cells were then washed twice and foci counted.
Soft agar assay
A total of 104 Panc02-GFP, Panc02-mTrop2 cells were plated in triplicate in 6-well plates with 2 ml of growth medium containing 0.35% agar and used to overlay 4 ml layers of growth medium containing 0.7% agar. Colonies with a diameter greater than 0.2 mm were counted using a dissecting microscope.
Mouse models
Subconfluent and stable Panc02-GFP and Panc02-mTrop2 cells were harvested and resuspended in DMEM. For the orthotopic murine model, Panc02 cells were also used. For the subcutaneous (s.c.) tumor model, 2 × 105 cells were inoculated into the right flank of 7- to 8-week-old female nude mice (National Cancer Institute-Charles River). For the orthotopic tumor model, 5 × 104 cells were injected into the pancreas of 7- to 8-week-old female nude mice. For intrapancreatic injection, mice were anesthetized with 2.5% Avertin and an incision of 1-cm was made in the left subcostal region. The spleen was exteriorized and tumor cells in a volume of 50 μl were injected into the pancreas. For the s.c. tumor model, tumor size was measured twice weekly using digital calipers and the tumor volume was calculated with the formula: tumor volume (mm3) = [length (mm)] × [width (mm)]2 × 0.52. For the orthotopic tumor model, mice were euthanized after 14 days. Tumors were extracted and weighed. All experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee at Baylor College of Medicine.
Statistical analysis
Quantitative results are shown as mean ± SD. Statistical analysis was done using Student's t tests for paired data between the control and mTrop2 groups or one-way ANOVA to determine significant difference between groups. P < 0.05 was considered significant.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
RC performed cell line generation, western blotting, proliferation assays, cell cycle analysis, wound healing assay, focus formation assay, flow cytometry, soft agar assay, AP-1 SEAP assays, immunohistochemistry, subcutaneous and orthotopic mouse models and drafted manuscript. SZ helped in the subcutaneous and orthotopic models. ML and CC participated in discussion. QY participated in discussion and manuscript preparation and provided grant support for this study. All authors read and approved the final version of this manuscript.