Serum depletion induced cancer stem cell-like phenotype due to nitric oxide synthesis in oncogenic HRas transformed cells

Abstract

Cancer cells rewire their metabolism and mitochondrial oxidative phosphorylation (OXPHOS) to promote proliferation and maintenance. Cancer cells use multiple adaptive mechanisms in response to a hypo-nutrient environment. However, little is known about how cancer mitochondria are involved in the ability of these cells to adapt to a hypo-nutrient environment. Oncogenic HRas leads to suppression of the mitochondrial oxygen consumption rate (OCR), but oxygen consumption is essential for tumorigenesis. We found that in oncogenic HRas transformed cells, serum depletion reversibly increased the OCR and membrane potential. Serum depletion promoted a cancer stem cell (CSC)-like phenotype, indicated by an increase in CSC markers expression and resistance to anticancer agents. We also found that nitric oxide (NO) synthesis was significantly induced after serum depletion and that NO donors modified the OCR. An NOS inhibitor, SEITU, inhibited the OCR and CSC gene expression. It also reduced anchorage-independent growth by promoting apoptosis. In summary, our data provide new molecular findings that serum depletion induces NO synthesis and promotes mitochondrial OXPHOS, leading to tumor progression and a CSC phenotype. These results suggest that mitochondrial OCR inhibitors can be used as therapy against CSC.

Keywords: HRas; OXPHOS; cancer stem cell; nitric oxide; serum depletion.

Figure 1. Tumorigenesis and mitochondrial respiratory function of HRASG12V-expressing wild type (WT) and p32 knockout (KO) MEF cells

A. Immunoblotting analysis of p32 (a mitochondrial RNA chaperone protein) and COX1 (a mitochondrial respiratory complex subunit) expression. B. Soft agar assay of WT or p32 KO MEF cells transfected with the control (Ctrl) vector or HRasG12V. After 2 weeks incubation, colonies larger than 20,000 μm² per 180 mm² were counted. The microscopic image and histogram show the colony numbers of each sample. Scale bar = 1 mm. C. Oxygen consumption ratio (OCR) of control and HRasG12V expressing MEF cells. OCR was measured by using an XFe24 analyzer. The histogram shows the basal respiration rate (Basal), ATP production rate (ATP) and maximal respiration rate (Maximal) calculated from the left line chart. Data show the mean ± SD of quadruplicate assays and *p < 0.05; control versus HRasG12V. D. Mitochondrial membrane potentials (MMP) using a TMRM probe were measured in HRASG12V-expressing MEF cells by FACS analysis. Unstained cells were used as a negative control. E. ROS production was measured in HRASG12V-expressing cells by using a MitoSOX probe and FACS analysis. F. OCR of WT MEF cell, control and HRasG12V-transfected p32 KO MEF cells. *p < 0.05; WT control versus p32 knockout control.

Figure 2. Serum depletion increases the mitochondrial respiratory function of HRASG12V-expressing MEF cells

A. OCRs were measured after serum depletion (0.1% FBS) for 12 hr. Data show the mean ± SD of quadruplicate assays and *p < 0.05; 10% FBS versus 0.1% FBS. B. MMP were measured after serum depletion for 12 hr of HRASG12V-expressing MEF cells by FACS analysis. C. ROS production was measured after serum depletion for 12 hr in HRASG12V-expressing MEF cells by using a MitoSOX probe and FACS analysis. D. MitoTracker Red staining of HRASG12V-expressing MEF cells incubated with different serum concentrations. Scale bar = 5 μm. The lower image is the enlarged region denoted in the upper image. E and F. Immunoblotting analysis of mitochondrial fission/fusion proteins (E) and mitochondrial respiratory subunit proteins (F) of HRASG12V-expressing MEF cells. Cells incubated with each medium for 12 hr were subjected to SDS-PAGE.

Figure 3. Serum depletion induced CSC gene expression and resistance to anti-cancer drugs

A. Oct4, Aldh1a1 and Cd133 gene expression were measured by RT-PCR analysis after serum depletion of HRASG12V-expressing MEF cells for 48 hr. Embryonic stem (ES) cells were used as a positive control. 18s rRNA was used as a loading control. B. Cell proliferation assay of HRASG12V-expressing MEF cells. Cells were treated with CPT11 (0.05μM) or VP16 (0.1μg/ml) with different FBS concentrations and durations (10% for 36 hr or 0.1% for 72 hr). The fold change in cell numbers was used as a measure of proliferation. Data show the mean ± SD of quadruplicate assays. C. MTS assay; cells treated with serum depletion for 24 hr were treated with various concentrations of CPT11 and Paclitaxel(PTX) for another 48 hr. Data were normalized to the control. *p < 0.05; 10% FBS versus 0.1%.

Figure 4. Serum depletion induced NO synthesis and iNos (Nos2) gene expression

A. Intracellular arginine level was measured by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Data show the mean ± SD of quadruplicate assays and *p < 0.05; 10% FBS versus 0.1%. B, C. FACS analysis shows intracellular NO production by using the DAF-FM DA probe in HRASG12V-expressing MEF cells after serum depletion for 30 min (B) or pretreated with the NO inhibitor SEITU (C) for 3 hr and then were subjected to serum depletion (0.1% FBS) for another 30 min. D. RT-PCR analysis of iNos (Nos2) gene expression in HRASG12V-expressing MEF cells incubated with serum depletion for 48 hr or pretreated with SEITU (250 μM) for 12 hr before serum depletion. 18s rRNA was used as a loading control. E. Cells pretreated with SEITU for 6 hr were incubated with 0.1% FBS for 12 hr. OCR were measured by using an XFe24 analyzer. Data show the mean ± SD of quadruplicate assays and *p < 0.05; 0.1% FBS vs 0.1% FBS + SEITU. F. OCR of HRASG12V-expressing MEF cells incubated with or without the NO donor S-nitroso-N-acetylpenicillamine (SNAP, 2 μM). SNAP was added 2 hr before the assay started under 10% serum conditions. *p< 0.05; 10% FBS vs 10% FBS + SNAP.

Figure 5. Signal transduction of wild type HRAS and HRASG12V-expressing MEF cells under serum-depleted conditions

Cells were incubated with different serum concentrations for 24 hr. Immunoblotting analysis of A. RAS pathway and glycolysis B. AMPK and mTOR pathway proteins.

Figure 6. Suppression of anchorage-independent growth effect of SEITU and metformin on HRASG12V-expressing MEF cells

A. Soft agar colony formation assay of HRASG12V-expressing MEF cells treated with metformin (1 mM), SEITU (250 μM) or not were performed under 10% or 1.5% serum conditions. Left panel is the microscopic image. Scale bar = 1 mm. The right histogram shows colony numbers of each sample. B. Immunoblotting analysis: HRASG12V-expressing MEF cells pretreated with SEITU (250 μM) or metformin (1 mM) for 12 hr were serum depleted for another 24 hr. C. RT-PCR analysis of gene expression level of cancer stem cell genes in HRASG12V-expressing MEF cells. Total RNA was isolated from HRASG12V-expressing MEF cells incubated with 10% or 0.1% FBS for 48 hr. SEITU (250 μM) was added 12 hr before serum depletion. D. Cells pretreated with SEITU (250 μM) or metformin (1 mM) for 12 hr were incubated under serum depletion for another 24 hr. E. OCR of HRASG12V-expressing cells treated with or without metformin (1 mM). Metformin was added 6 hr before serum depletion. The serum concentration was 0.1%. Data show the mean ± SD of quadruplicate assays and *p < 0.05; 0.1% FBS versus 0.1% FBS + metformin.

Figure 7. Serum depletion induced NO synthesis and CSC features

Oncogenic HRas led to suppression of the mitochondrial oxygen consumption rate (OCR) (Warburg effect). In the oncogenic HRas transformed cells, serum depletion reversibly increased the OCR, NO synthesis and CSC features. Metformin (anti-diabetic drug) and SEITU (NOS inhibitor) suppress OXPHOS, stem cell gene expression and tumor progression.