Hypoxia is a hallmark of progressive cancer. Hypoxic cancer cells trigger glycolysis in response to a decreased O2 supply to meet metabolic and bioenergetic demands. Meanwhile, these responses to hypoxia and alterations of the microenvironment promote cancer cell metastasis by increasing transcription of hypoxia-inducible factor (HIF)-regulated genes. However, the detailed mechanism by which hypoxia regulates cancer cell metastasis and glycolysis remains to be investigated. In the present study, we identified that metadherin (MTDH), a multifaceted oncogene, is involved in the regulation of head and neck squamous cell carcinoma (HNSCC) metastasis and invasion under hypoxic conditions. Furthermore, the study indicated that there is a positive feedback loop between HIF-1α and MTDH in HNSCC cells, and that hypoxia promotes HNSCC cell metastasis and epithelial-mesenchymal transition by mediating the HIF-1α-MTDH loop. These findings implicate HIF-1α-MTDH as a promising target for anticancer drugs in solid tumors, and help to explain the pro-tumorigenic and unfavorable effect of MTDH on HNSCC observed in our previous studies.
Head and neck squamous cell carcinoma (HNSCC) affects ~500,000 new patients annually worldwide (
Recent evidence has indicated that microenvironmental constituents in the cancer stroma substantially influence the propensity of cancer cells to metastasize (
Metadherin (MTDH), also known as AEG-1 or Lyric, is located on human chromosome 8q22 and is recognized as an oncogene that regulates numerous signaling pathways in cancer cells (
In the present study, we confirmed that hypoxia increased MTDH expression via HIF-1α expression. Knockdown of MTDH expression in HNSCC cell lines interrupted hypoxia-induced metastasis and glycolysis. Furthermore, reduced MTDH expression decreased HIF-1α expression. The present study indicated that there is a positive feedback loop between MTDH and HIF-1α in HNSCC. Hypoxia promotes HNSCC metastasis and glycolysis via an MTDH-HIF-1α loop pathway.
The Tu686 cell line was provided by Dr Zhuo Chen of Emory University's Winship Cancer Institute in Atlanta, Georgia and was maintained as monolayer cultures in Dulbecco's modified Eagle's medium (DMEM)/F12 medium (1:1) supplemented with 10% fetal bovine serum (FBS) at 37°C under normoxia in a modular incubator chamber, or under hypoxia with 5% CO2 and 1% O2 balanced with N2. The medium was changed every other day. Exponentially growing cells were used for the following experiments.
MTDH cDNA (GeneCopoeia, Guangzhou, China), shRNA (sc-77797V; Santa Cruz Biotechnology, Santa Cruz, CA, USA, CA, USA), HIF-1α cDNA (OriGene Technologies, Inc., Rockville, MD, USA), siRNA (sc-35561; Santa Cruz Biotechnology), and their corresponding control plasmids were transfected into the Tu686 cell line according to the manufacturers instructions.
All western blot analysis was performed as previously described (
Primers for β-actin, HIF-1α, E-cadherin, vimentin, N-cadherin and VEGF mRNA were synthesized and validated by Beijing Sunbiotech Co., Ltd. (Beijing, China). TRIzol® reagent (cat no. 15596026; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used for RNA preparation. PowerUp™ SYBR®-Green Master Mix (A25776; Thermo Fisher Scientific) was used in the PCR amplification. The expression level was quantified by an Applied Biosystems device using the 2−ΔΔCt method. Melting curve analysis was performed at the end of the amplification cycle to verify non-specific amplification. The detailed information of PCR primers is listed as follows: VEGF-L, 5′-aggccagcacata ggagaga-3′ and VEGF-A, 5′-tttcttgcgctttcgttttt-3′; HIF-1α-L, 5′-ccacctatgacctgcttggt-3′ and HIF-1α-R, 5′-tatccaggctgtgt cgactg-3′; β-actin-L, 5′-ctcttccagccttccttcct-3′ and β-actin-R, 5′-agcactgtgttggcgtacag-3′; E-cadherin-L, 5′-tgcccagaaaa tgaaaaagg-3′ and E-cadherin-R, 5′-gtgtatgtggcaatgcgttc-3′; vimentin-L, 5′-gagaactttgccgttgaagc-3′ and vimentin-R, 5′-tcca gcagcttcctgtaggt-3′; N-cadherin-L, 5′-aggatcaaccccatacacca-3′ and N-cadherin-R, 5′-tggtttgaccacggtgacta-3′.
Cells seeded into 6-well plates were allowed to proliferate to near 100% confluence and were wounded by removing a line of cells using a disinfected Eppendorf tip (100 µl). After washing with FBS-free medium (0 h), the first image was photographed under a microscope. The second image was captured in the same way after 48 h. The closure of wound width in images was measured three times by Photoshop software. The wound healing rate = (average wound width at 0 h) - (average wound width at 48 h)/(average wound width at 0 h) × 100%.
Transfected cells were plated in Transwell cell culture inserts (Corning Costar, Corning, NY, USA) at a density of 2×104 cells/well. The cells were maintained and allowed to migrate for 48 h, after which the cells that had not invaded were removed from the upper surface using a cotton swab. The cells that had invaded to the lower surface were stained with crystal violet solution and photographed under a microscope.
Glucose Assay kit (ab65333), Deproteinizing Sample Preparation kit-TCA (ab204708), Glucose Uptake Assay kit (ab136955) and L-lactate assay kit (ab65330; all from Abcam, Cambridge, MA, USA) were utilized in the glycolysis-related assays according to the manufacturers protocols.
All statistical analyses were performed using IBM SPSS statistical software, version 21.0 (SPSS, Inc., Chicago, IL, USA). The differences between data derived from two groups in the experiments were statistically analyzed by a Student's test. P<0.05 was considered to indicate a statistical significant result. All tests were two-sided.
Hypoxia is a hallmark of cancer cells. Thus, we investigated whether hypoxia induces cell migration and invasion in the HNSCC Tu686 cell line. The wound-healing rate indicated that hypoxia significantly promoted Tu686 cell healing after 48 h in hypoxia compared to cells cultured in normoxia (P<0.05;
Since the migration and invasion of HNSCC cells were increased by hypoxic treatment, qPCR and western blotting were applied to assess whether hypoxia regulates the expression of VEGF and EMT biomarkers, which are key molecules involved in cell metastasis. VEGF mRNA was promoted in the hypoxic HNSCC cells. The mRNA and protein expression levels of vimentin and N-cadherin were upregulated, while E-cadherin protein was decreased following hypoxic treatment (
High expression of MTDH protein was previously shown to predict poor prognosis in laryngeal squamous cell carcinoma patients (
As the aforementioned results indicated that hypoxia regulated MTDH expression, we subsequently investigated whether HIF-1α, a classic indicator of hypoxia, regulated MTDH expression in Tu686 cells. MTDH expression was assessed in cells transfected with HIF-1α cDNA and siRNA.
The expression level of MTDH protein was markedly decreased when HIF-1α was knocked down in the Tu686 cell line under hypoxic and normoxic conditions (
Our previous studies showed that MTDH regulated HNSCC metastasis by altering the expression of EMT proteins and VEGF in normoxic cell culture (
In the present study, we confirmed that hypoxia induced cell metastasis, glycolysis and MTDH expression in an HNSCC cell line. Additionally, downregulation of MTDH expression could offset the hypoxia-induced increase in HNSCC cell metastasis and glycolysis. We also verified that HIF-1α was crucial for the regulation of MTDH by hypoxia. Importantly, MTDH was found to regulate HIF-1α expression in turn. Furthermore, both MTDH and HIF-1α could partially reverse the hypoxia-induced increases in EMT biomarkers and VEGF expression in HNSCC cells.
A range of cellular signaling pathways, relating to angiogenesis, autophagy, EMT and energy metabolism, are activated in cells in order to adapt to hypoxic stress (
When cells are cultured in an environment of 1% O2, HIF-1α protein is stabilized by a reduction in ubiquitination-mediated degradation (
The present study showed that hypoxia increased the mRNA expression levels of glycolysis-related genes, such as MCT1, MCT4, GLUT1, HK2, PGI, PGK1, ENO1, PFK2 and LDHA, in HNSCC cells (data not shown). In addition, our data showed that hypoxia promoted glucose uptake, lactate production and cell invasion in HNSCC cells. In the clinical setting, glucose uptake has been used in PET-CT imaging for assessing tumor metastasis and relapse (
Notably, the present study indicated that MTDH protein expression was elevated in hypoxic HNSCC cells. MTDH is well-documented as an oncoprotein in human malignancies, including lung, colon, breast, live, glioma cancer and HNSCC (
The multifaceted roles of MTDH in cancer are due to the diverse downstream cellular signals activated, including the PI3K/AKT, NF-κB, Wnt/β-catenin and MAPK pathways (
The mutual regulation of MTDH and HIF-1α identified in the present study may indicate a feedback loop connection between MTDH and HIF-1α in HNSCC. Additionally, it is possible that the intermodulation of MTDH and HIF-1α may affect cell metastasis and EMT together. The present investigation indicated that EMT biomarkers and cell metastasis were enhanced significantly in hypoxic HNSCC cells. When MTDH shRNA was transfected into hypoxic cells (and HIF-1α was also activated), EMT biomarker expression and cell metastasis were reduced, suggesting that disruption of the MTDH-HIF-1α loop connection inhibited HNSCC cell metastasis and increased its resistance to EMT. There are two possible mechanisms: HIF-1α may activate the EMT transcription factor Twist in hypoxic HNSCC cells (
Considering the aforementioned findings in HNSCC, there may be a loop connection between MTDH and HIF-1α in HNSCC cells, and hypoxia may promote HNSCC cell glycolysis and metastasis via this loop. If cells grow at a sufficient distance from the oxygen-delivering vasculature within HNSCC tumors, adaption to hypoxic stress occurs, resulting in the nuclear translocation of HIF-1α to promote the transcription of glycolytic genes and oncogenes, including LDHA and MTDH, to accelerate energy synthesis and progression, respectively. MTDH activates the PI3K and NF-κB pathways, which may increase HIF-1α expression in turn and further enhance the aforementioned process. The present study also found that HNSCC cell migration/invasion and glycolysis in hypoxia were suppressed when this positive feedback was disrupted by knockdown of MTDH expression. Thus, targeting MTDH in HNSCC may be a promising direction for improving the survival time of HNSCC patients, as MTDH reduced the malignant activity of HNSCC cells in normoxia and hypoxia. In summary, hypoxia promotes HNSCC cell migration/invasion and glycolysis by mediating a HIF-1α-MTDH feedback loop. These findings implicate HIF-1α-MTDH as a promising target for anticancer drugs in solid tumors, and help to explain the pro-tumorigenic and unfavorable effect of MTDH in HNSCC as reported in our previous study.
We thank all our laboratory members for their insightful suggestions in discussion. The present study was funded by grants from the National Natural Science Foundation of China (nos. 81602389, 81472696, 81202128 and 81272974), and the Natural Science Foundation of Hunan Province (nos. 2017JJ3456 and 2015JJ3137).
Hypoxia induces HNSCC metastasis and glycolysis in the Tu686 cell line. (A) Wound healing experiment showed that there was a different rate of migration between normoxic and hypoxic cells. (B) Quantitative analysis revealed that the percentage of wound healing in hypoxic Tu686 cells was significantly higher than that in the normoxic Tu686 cells (82.0±4.0 vs. 59.3±7.6%; *P<0.05). (C) Representative figures for evaluation of cell invasion in hypoxic and normoxic Tu686 cells. (D) Quantitative analysis showed that hypoxia induced Tu686 cell invasion (157±7 vs. 96±13; **P<0.01). (E and F) Glucose uptake and lactate production were significantly promoted in the hypoxic Tu686 cells after 24 and 48 h; **P<0.01. (G) Amount of glucose in the supernatant was decreased in hypoxic Tu686 cells after 24 and 48 h; **P<0.01..
Hypoxia induces EMT and MTDH expression in HNSCC cells. (A) mRNA expression of E-cadherin, N-cadherin, vimentin and VEGF was compared in hypoxic and normoxic Tu686 cells (**P<0.01). (B) Hypoxia induced MTDH protein expression and EMT. β-actin was used as an internal reference.
MTDH reverses HNSCC metastasis and glycolysis induced by hypoxia in the Tu686 cell line. (A) The percentage of wound closure in hypoxic cells with MTDH knockdown was approximately equal to that noted in normoxic cells. (B) Quantitative analysis revealed there was no significant difference in wound closure percentage in normoxic Tu686 and hypoxic Tu686 cells with MTDH knockdown (60.0±7.0 vs. 65.3±8.5%, respectively; NS indicates P>0.05). Meanwhile, the percentage of wound closure in hypoxic cells was significantly higher than both of normoxic cells and hypoxic cells with MTDH downregulation (84.0±6.2 vs. 60.0±7.0 and 65.3±8.5%, respectively; *P<0.05). (C) The number of invading cells in hypoxic cells with MTDH knockdown was approximately equal to the number of normoxic cells. (D) There was no significant difference in invading cell number in normoxic Tu686 and hypoxic Tu686 cells with MTDH knockdown (92±13 vs. 123±14 respectively; NS indicates P>0.05). The invading cell number in hypoxic cells was significantly higher than both of normoxic cells and hypoxic cells with MDH downregulation (188±13 vs. 92±13 and 123±14, respectively; **P<0.01). (E and F) The promotion of glucose uptake and lactate production by hypoxia were significantly reversed by knockdown of MTDH expression. (G) The amount of glucose in the supernatant of hypoxic cells was increased to the level of nomoxic cells. (H) Western blotting showed that the MTDH protein was significantly decreased in the cells with MTDH shRNA transfection.
Mutual regulation of HIF-1α and MTDH in Tu686 cells. (A) MTDH protein expression was decreased when HIF-1α siRNA was transfected in both normoxic and hypoxic cells. (B) Compared to Tu686 cells transfected with the plasmid vector, MTDH protein expression was upregulated in Tu686 cells with HIF-1α cDNA transfection. (C) HIF-1α mRNA was significantly upregulated in MTDH-overexpressing cells (**P<0.01). (D) Western blotting indicated that MTDH protein was significantly increased in the cells transfected with MTDH cDNA. In addition, HIF-1α protein was promoted in the MTDH-overexpressing cells.
MTDH and HIF-1α regulate EMT-related biomarkers and VEGF expression in Tu686 cells. (A) mRNA expression of E-cadherin, N-cadherin, vimentin and VEGF was compared in normoxic Tu686, hypoxic Tu686 and hypoxic Tu686 cells with MTDH knockdown (*P<0.05, **P<0.01). (B) HIF-1α induced the expression of N-cadherin, vimentin and VEGF mRNA (**P<0.01). (C) The expression of N-cadherin, vimentin and VEGF mRNA were decreased by HIF-1α knockdown in normoxic and hypoxic Tu686 cells (*P<0.05, **P<0.01). NS indicates P>0.05.