Background
In the early twentieth century Otto Warburg recognized a metabolic phenomenon that occurred in cancer cells, currently known as the Warburg effect [
1‐
4]. Warburg discovered cancer cells favor glycolysis rather than oxidative phosphorylation for energy production, even in the presence of oxygen [
1‐
4]. It was originally hypothesized that irreversible injury of mitochondrial respiration is the cause of cancer cell formation [
1]. However, this hypothesis was to some extent discredited as most cancers retain their ability to exploit mitochondrial respiration, although to a lesser degree than normal cells [
5‐
7]. As a result of unrestricted glycolysis the tumor microenvironment is spatially acidic [
8‐
10]. Extracellular acidosis has pleiotropic effects on tumor growth and cancer progression [
10‐
14]. Tumor acidosis can stimulate cell death, reduce cell proliferation, and induce chromosomal instability of normal somatic cells and cancer cells [
13‐
16]. In addition, tumor cells that become resistant to extracellular acidosis have been reported more malignant and invasive [
17,
18]. Therefore, extracellular tumor acidosis augments cancer progression in a Darwinian manner worsening disease prognosis. As such, it is imperative to understand how tumor cells sense and respond to acidic surroundings for adequate comprehension of cancer development.
T cell death-associated gene 8 (TDAG8, also known as GPR65) is a member of the proton sensing G-protein coupled receptor family, which also includes GPR4, GPR68 (OGR1), and GPR132 (G2A). The family of G-protein coupled receptors is activated by extracellular acidosis, which illuminates a receptor signaling connection to the acidic conditions found in tumor microenvironment [
19]. The human TDAG8 gene has been mapped to chromosome 14q31-32.1, a location that abnormalities associated with T cell lymphoma and leukemia are found and is primarily expressed in immune cells and leukocyte-rich tissues such as circulating peripheral leukocytes, spleen, thymus, and tonsils [
20,
21]. TDAG8 has been reported to have both pro- and anti-oncogenic effects in blood cancers, which indicate TDAG8 effects are cell type and context dependent [
22‐
24]. Therefore, it is imperative to understand the contextual effects of TDAG8 in hematological malignancies. Blood cancer cells are generally glycolytic and produce lactic acid that can acidify the microenvironment. Some types of hematological malignancies, such as lymphomas, have the ability to form solid tumors in which the tumor microenvironment is acidic. Other types of hematological malignancies, such as leukemia and multiple myeloma, form in and metastasize to bone marrow that has hypoxic and possibly acidic niches [
25,
26]. In rare cases, systemic lactic acidosis occurs in patients with hematological malignancies and is associated with poor prognosis [
27].
In this study the effects of acidosis and TDAG8 gene expression was investigated in blood cancers. TDAG8 gene expression was examined in hematological malignancies revealing a significant reduction in comparison to normal immune cells and leukocyte-rich tissues. Functional studies demonstrated that restoration of TDAG8 gene expression suppressed the growth, migration and metastasis of blood cancer cells and sensitized them to extracellular acidosis.
Methods
TDAG8 gene expression was investigated in blood cancers using the Oncomine database. A differential analysis of TDAG8 gene expression was performed between leukemia, lymphoma, and myeloma datasets and normal immune cells and leukocyte-rich tissue. Additional bioinformatics analyses were performed on datasets from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO). The raw data was downloaded using expression console software HG_u133_Plus_2 and HG_u133a_2 as libraries. The analysis was run and the RMA normalization method was used to generate the values graphed. The output file was then merged with the probe set downloaded from Affymetrix.
Plasmid constructs
The MSCV-huTDAG8-IRES-GFP construct was made previously and the empty construct, MSCV-IRES-GFP, was used as a control [
23]. For the pMSCV-huTDAG8-IRES-YFP-P2A-OF-LUC construct, the open reading frame of human TDAG8 was amplified using primers designed to contain the EcoRI and XhoI restriction enzyme sites: 5′-ATAAGAAT
GAA TTCACCATGAACAGCACATGTATTGAAGAA-3′ and 5′-ATAAGAATGAATTC
CTCGAG CTACTCAAGGACCTCTAATTCCAT-3′. The pMSCV-IRES-YFP-P2A-OF-LUC plasmid was then digested with EcoRI and XhoI and the huTDAG8 open reading frame was cloned into it generating the pMSCV-huTDAG8-IRES-YFP-P2A-OF-LUC construct. The resultant construct was verified by DNA sequencing.
Cell lines and culture
All the cancer cell lines were obtained from American Type Culture Collection (ATCC) with characterization as described in the product sheet. All cell lines were cultured in Roswell Park Memorial Institute medium (RPMI) supplemented with 10% fetal bovine serum (FBS) in a tissue culture incubator set at 37 °C and 5% CO
2. RPMI/FBS medium was buffered using 7.5 mM HEPES, 7.5 mM EPPS and 7.5 mM MES, known collectively as RPMI/HEM, as previously described [
23]. Cells used for experiments were > 95% viable as assessed by the trypan blue dye exclusion method.
To generate green fluorescent protein (GFP) and yellow fluorescent protein (YFP)/luciferase (Luc) cell lines with restored TDAG8 gene expression, retroviral-mediated cell transduction was performed as previously described [
28]. To generate the Gα13 signaling-deficient cell lines the p115 Rho RGS construct was subcloned into the pQCXIP vector as previously described [
28,
29]. The p115 Rho RGS-pQCXIP and empty pQCXIP retroviral vectors were then stably transduced into U937 cells.
EdU cell proliferation assay
The Click-iT® Plus EdU Imaging Kit (Thermo Fisher Scientific) was used to examine U937/Vector and U937/TDAG8 cell proliferation. Fluorescence microscopy with the EVOS®
fl Digital Inverted Microscope was used to take images of cells incorporating Hoescht® 33,342 dye and the EdU analogue, and also images with transmitted light in the same field of view at a 200 × magnification. Adobe Photoshop’s counting tool was used to count the number of total cells according to Hoescht® 33,342 dye and proliferating cells according to EdU positive cells.
Cell growth competition assay
The same number of non-GFP expressing cells (U937/Parental or RPMI 8226/Parental) and GFP expressing cells (Vector or TDAG8) were mixed into a well and the percentages of non-GFP and GFP cells were measured using flow cytometry over 14 days. Cell population percentages were analyzed and graphed.
Annexin V/7AAD staining
Cells were stained with the PE Annexin V Apoptosis Detection Kit I (BD Biosciences). Emission of single cell fluorescence was measured at 572 nm for Annexin V, 647 nm for 7AAD, and 525 nm for GFP. The results were analyzed with CellQuest software (BD Biosciences).
Quantitative reverse transcription-polymerase chain reaction (RT-PCR)
Gene expression was determined by quantitative RT-PCR as previously described [
23], using the following TaqMan predesigned primer/probes from Invitrogen, TDAG8 (Hs00269247_s1), c-myc (Hs00153408_m1), and β-actin (Hs99999903_m1). Relative gene expression was calculated using the 2
−ΔΔCt method [
30].
Western blotting
Western blot was performed as previously described [
23]. Antibodies of c-myc (product #5605), phospho-paxillin (Y118) (#2541), phospho-CREB (S133) (#9198), CREB (#9197), caspase-3 (#9665), caspase-9 (#9502), cleaved PARP XP (#5625), and β-actin (#4970) were purchased from Cell Signaling Technology.
Histology
Immunohistochemistry (IHC) for antibodies against c-myc (Abcam, #ab32072), cleaved PARP (Cell Signaling Technology, #5625), Ki67 (Abcam, #ab15580), and human nucleoli (Abcam, #ab190710) was performed on paraffin-embedded 5 µm sections. Antigen retrieval was performed by boiling slides in TRIS–EDTA + 0.1% Tween-20 pH 9.0 antigen retrieval buffer for 10–18 min. SuperPicture™ 3rd Gen IHC Detection Kit (Invitrogen) was used to complete immunohistochemistry.
Transwell cell migration assays
Two hundred microliter of cells were seeded into the transwell insert at 5 × 106 cells/ml in migration buffer consisting of RPMI media buffered to pH 7.4 and pH 6.4 without FBS and supplemented with 0.1% BSA. Chemoattractant (5 ng/mL SDF1-α) was then added into the bottom well. The plates were then incubated at 37 °C and 5% CO2 for 5 h. After 5 h the number of migrated cells was quantified using flow cytometry.
Cell attachment assays
Matrigel solution was added into each well to form a thin layer of gel. U937 cells were plated onto Matrigel and incubated at 37 °C and 5% CO2 for 1 h in culture media buffered to pH 7.4 or 6.4. After 1 h the media was aspirated and the wells were washed 4 times with RPMI to remove non-adherent cells. For the endothelial cell attachment assay Human Umbilical Vein Endothelial cells (HUVEC) were seeded on 0.1% gelatin-coated plates to create a monolayer. Next, U937 cells were plated in each well in culture media buffered to pH 7.4 or 6.4. Plates were incubated at 37 °C and 5% CO2 for 1 h. After 1 h the media was aspirated and the wells were washed 4 times with RPMI. For all attachment assays several images were taken in various areas of the well to give an adequate representation. Cell attachment was quantified by counting every cell in each field of view.
NOD.CB17-Prkdc<SCID>/J mice were purchased from Jackson Laboratories and bred at East Carolina University animal facilities for research purposes. All experimental procedures were performed according to the NIH guide for the care and use of laboratory animals and the institutional regulations. In each experiment, 5 × 106 U937 or Ramos cells were injected into the flanks of SCID mice. Tumor growth was measured daily using a caliper. Tail vein injections were performed with 2 × 106 U937 cells in SCID mice. When mice reached endpoint parameters, e.g. lethargy, hunched posture, and unkempt appearance, they were euthanized for analysis. The experiment was terminated 8 months after injection when the remaining mice showed no signs of disease progression. To monitor U937-Luc tumor growth in vivo, intraperitoneal injection with d-Luciferin (Caliper Life Sciences, Catalogue # 119222) at 15 mg/ml in DPBS was performed. Subsequently, mice were imaged using the IVIS Lumina XR unit.
Statistical analysis
Statistical analysis was performed using the GraphPad Prism software. The unpaired t test was used to test for statistical significance between 2 groups. Error bars represent ± standard error (SEM) in all graphs. ns: P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.
Discussion
The effect of extracellular acidosis on cancer progression is complex; therefore it is important to understand the cancer cell biological response to it [
13]. In this study it was determined that diverse extremities of extracellular acidosis had differential effects on cellular proliferation (Fig.
2c–e, g, h). It was clearly demonstrated that mild extracellular acidosis, pH 6.9, increased U937 cell proliferation while severe acidosis, pH 6.4, repressed it (Fig.
2c). In addition, this report evaluated the effects of tumor acidosis on c-myc oncogene expression in vivo. In tumor sections nearest necrotic regions, known to be acidic [
17], c-myc oncogene expression was reduced significantly in live tumor cells (Fig.
3d–i). However, invasive cancer cells that were invading new tissue, devoid of blood vessels and nutrients, did not follow this pattern (Fig.
3d–i). Conversely, invasive tumor cells had increased expression of c-myc and Ki-67 signifying they are actively proliferating and invasive despite extracellular acidosis and nutrient deprivation (Fig.
3d–i). The discovered pattern of tumor cell invasiveness in harsh extracellular conditions concurrently associating with increased cell proliferative markers is similar to an acid resistant phenotype described previously [
17,
18]. Importantly, understanding that extracellular acidosis reduces c-myc oncogene expression in some tumor cells while not in others provides an original understanding for the heterotypic and spatial tumor cell response to extracellular acidosis. To understand how tumor cells sense extracellular acidosis the proton sensing G-protein coupled receptor TDAG8 was investigated.
The data provided in this study indicates that TDAG8 gene expression is suppressive for U937 cell malignancy. It was discovered that TDAG8 gene expression is reduced in the majority of blood cancers in comparison to normal immune cells or leukocyte-rich tissue (Table
1 and Fig.
1). In addition, higher expression of the TDAG8 gene correlates with increased sensitivity toward various chemotherapeutics (Table
2). Using U937 acute myeloid leukemia cells as a model system TDAG8 gene expression was restored to a normal physiological level to test the hypothesis that TDAG8 gene expression provides a disadvantage for blood cancer cell malignancy. Restoring TDAG8 gene expression in U937 cells reduced cell proliferation in vitro and tumor growth and metastasis in vivo (Figs.
2,
3,
6). The ability for TDAG8 gene expression to provide a disadvantage for U937 cell proliferation, tumor growth, and metastasis confirmed that TDAG8 gene expression provides a disadvantage for blood cancer progression. Similar tumor suppressive functions of TDAG8 were observed in other blood cancer cells such as Ramos Burkitt lymphoma and RPMI 8226 multiple myeloma cells (Additional file
1: Figures S3 and S5). Moreover, TDAG8’s inherent ability to reduce c-myc expression at physiological pH 7.4 and acidic pH 6.4 is central (Fig.
4a–c) [
23]. This is consistent with previous results demonstrating that TDAG8 exhibits constitutive activity at physiological pH and is further activated at acidic pH [
41]. Moreover, the results from this report indicate that the inhibitory effects of acidosis on c-myc oncogene expression are partially due to TDAG8-mediated Gα13/Rho signaling (Fig.
4e–f). This idea aligns with recent reports that demonstrate Gα13/Rho signaling suppresses oncogenesis and acts as a tumor suppressor in lymphoma [
51‐
53]. Overall, it is hypothesized in this report that extracellular acidosis provides a selective pressure against cancer cells, which modulates clonal cell evolution. Moreover, TDAG8 is a proton sensor that plays an important role in this process, which has important implications for blood cancer progression as well as cancer cell clonal evolution parallel to extracellular acidosis found within the tumor microenvironment.
TDAG8 has been reported to have a diverse repertoire of pro- and anti-oncogenic effects that are cancer type and context dependent [
22‐
24,
54‐
57]. These seemingly conflicting observations can potentially be explained by TDAG8 downstream signaling—the Gα13 G-protein/Rho GTPase signaling. Studies have shown that Gα13 and Rho GTPases can have pro-oncogenic or anti-oncogenic effects in a cancer type and context dependent manner [
51‐
53,
58‐
61]. Gα13 and Rho GTPases exhibit pro-tumorigenic effects in various types of epithelial cancers [
58‐
60], but have anti-tumorigenic effects in hematological malignancies [
51‐
53]. The differential effects of Gα13/Rho GTPases may contribute to the anti-oncogenic effects of TDAG8 in hematological malignancies and the pro-oncogenic effects in other cancer types [
22‐
24,
54‐
57]. To better understand the involvement of TDAG8 in various tumor types, we performed additional bioinformatic analyses of the Oncomine database to evaluate TDAG8 (GPR65) expression in cancerous and normal tissues [
62‐
69]. The results show that TDAG8 is over-expressed in several types of epithelial tumors, including brain tumor, kidney cancer, and head and neck cancer when compared to their normal tissue counterparts (Additional file
2: Table S1). Methodologically, however, cautions should be taken when interpreting the expression profile of TDAG8 in epithelial tumors. RNA for gene expression analysis was isolated from whole tumor tissues [
62‐
69]. As a result, it is unclear whether the over-expression of TDAG8 is derived from epithelial cancer cells or from infiltrated leukocytes which are known to highly express TDAG8 [
20,
21,
70,
71]. Also, interestingly, TDAG8 expression is either not changed [
54] or down-regulated in lung cancer samples when compared to normal lung tissues (Additional file
2: Table S1). Taken together, the expression pattern of TDAG8 in epithelial tumors varies, probably complicated by infiltrated leukocytes that highly express TDAG8 [
20,
21,
70,
71]. In contrast, TDAG8 expression is consistently down-regulated in hematological malignancies when compared to normal blood cells and tissues (Table
1 and Fig.
1). Moreover, functional results from this study, together with previous studies [
22,
23], suggest that TDAG8 acts as a contextual tumor suppressor in hematological malignancies.
Authors’ contributions
CRJ and LVY conceptualized and designed the study; CRJ, EJS, LD, and KL performed the experiments; LVY supervised the study; CRJ, EJS, and LVY analyzed the data; TS and JTC performed the GEO microarray dataset analysis; CRJ wrote the first draft of the manuscript; LVY critically revised the manuscript. All authors read and approved the final manuscript.