34th Annual Meeting & Pre-Conference Programs of the Society for Immunotherapy of Cancer (SITC 2019): part 2

Document type: Articl

Reduced Warburg Effect in Cancer Cells Undergoing Autophagy:Steady-State H-1-MRS and Real-Time Hyperpolarized C-13-MRS Studies

Autophagy is a highly regulated, energy dependent cellular process where proteins, organelles and cytoplasm are sequestered in autophagosomes and digested to sustain cellular homeostasis. We hypothesized that during autophagy induced in cancer cells by i) starvation through serum and amino acid deprivation or ii) treatment with PI-103, a class I PI3K/mTOR inhibitor, glycolytic metabolism would be affected, reducing flux to lactate, and that this effect may be reversible. We probed metabolism during autophagy in colorectal HT29 and HCT116 Bax knock-out cells using hyperpolarized (13)C-magnetic resonance spectroscopy (MRS) and steady-state (1)H-MRS. 24 hr PI103-treatment or starvation caused significant reduction in the apparent forward rate constant (k(PL)) for pyruvate to lactate exchange compared with controls in HT29 (100 μM PI-103: 82%, p = 0.05) and HCT116 Bax-ko cells (10 μM PI-103: 53%, p = 0.05; 20 μM PI-103: 42%, p<0.0001; starvation: 52%, p<0.001), associated with reduced lactate excretion and intracellular lactate in all cases, and unchanged lactate dehydrogenase (LDH) activity and increased NAD+/NADH ratio following PI103 treatment or decreased LDH activity and unchanged NAD+/NADH ratio following starvation. After 48 hr recovery from PI103 treatment, k(PL) remained below control levels in HT29 cells (74%, p = 0.02), and increased above treated values, but remained below 24 hr vehicle-treated control levels in HCT116 Bax-ko cells (65%, p = 0.004) both were accompanied by sustained reduction in lactate excretion, recovery of NAD+/NADH ratio and intracellular lactate. Following recovery from starvation, k(PL) was significantly higher than 24 hr vehicle-treated controls (140%, p = 0.05), associated with increased LDH activity and total cellular NAD(H). Changes in k(PL) and cellular and excreted lactate provided measureable indicators of the major metabolic processes accompanying starvation- and drug-induced autophagy. The changes are reversible, returning towards and exceeding control values on cellular recovery, which potentially identifies resistance. k(PL) (hyperpolarized (13)C-MRS) and lactate ((1)H-MRS) provide useful biomarkers for the autophagic process, enabling non-invasive monitoring of the Warburg effect

Cellular characterization of starvation- and PI-103-induced autophagic cells.

<p>(a) Annexin V and propidium iodide staining (PI) assay measuring the proportion of cells undergoing apoptosis and necrosis in HT29 cells following 24 hr of 100 μM PI-103 treatment and HCT116 Bax-ko cells following 24 hr of 10 μM or 20 μM PI-103 treatment, or 6 hr or 24 hr of starvation. Data are expressed as means of percentage ± s.e.m. (minimum <i>n</i> = 3 in each group). Statistically significant changes are indicated (*p<0.05). (b) Cell number compared with 24 hr DMSO-treated controls in HT29 cells following 24 hr DMSO-treated recovery, 24 hr of 100 μM PI-103 treatment and its 48 hr recovery and 24 hr vehicle-treated controls in HCT116 Bax-ko cells following 24 hr of 10 μM or 20 μM PI-103 treatment, or 6 hr or 24 hr of starvation and its recovery from 24 hr of 20 μM PI-103 treatment or starvation. Data are expressed as means of percentage ± s.e.m. (minimum <i>n</i> = 3 in each group). Statistically significant changes are indicated (*p<0.05). (c) Cell cycle analysis. Data are means of percentage ± s.e.m. HT29 cells 24 hr DMSO-treated control, 24 hr DMSO-recovered control, 24 hr PI-103-treated 100 μM, 24 hr PI-103-recovered, 48 hr PI-103-recovered, (<i>n</i> = 3 in each group); HCT116 Bax-ko cells 24 hr DMSO-treated control, 24 hr DMSO-recovered control, 24 hr PI-103-treated 20 μM, 24 hr PI-103-recovered, 48 hr PI-103-recovered, (<i>n</i> = 3 in each group); HCT116 Bax-ko cells 6 hr full media control, 6 hr starvation (HBSS), 24 hr full media control, 24 hr starvation (HBSS), 48 hr HBSS-recovered (<i>n</i> = 3 in each group). *P<0.01.</p

Autophagy is induced in HT29 and HCT116 Bax-ko cells and is reversed after treatment ends.

<p>(a) Western blots of LC3, Cleaved PARP, Caspase 3 and α-tubulin in control HCT116 Bax-ko cells and in 10 μM or 20 μM PI-103-treated or 6 hr and 24 hr HBSS-treated HCT116 Bax-ko cells. (b) Western blots of LC3, Cleaved PARP, Caspase 3 and α-tubulin in control HT29 cells and in 24 hr 100 μM PI-103-treated HT29 cells and at 24 hr, 48 hr and 72 hr of their recovery. TRAIL-treated HCT116 WT cells are used as positive control for apoptosis, as TRAIL has been reported to induce apoptosis in HCT116 WT cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092645#pone.0092645-Han1" target="_blank">[22]</a>. As expected, the presence of cleaved PARP and highly reduced caspase 3 expressions were observed in 24 hr TRAIL-treated HCT116 WT cells when compared with vehicle-treated controls. (c) Western blots of LC3, Cleaved PARP, Caspase 3 and α-tubulin in control HCT116 Bax-ko cells and in 24 hr 20 μM PI-103-treated HCT116 Bax-ko cells and at 24 hr and 48 hr of their recovery. TRAIL-treated HCT116 WT cells are used as positive control for apoptosis. (d) Western blots of LC3, Cleaved PARP, Caspase 3 and α-tubulin in control HCT116 Bax-ko cells and in 24 hr of HBSS-treated HCT116 Bax-ko cells and at 24 and 48 hr of their recovery.</p

Assessment of apparent ¹³C-pyruvate to lactate exchange rate.

<p>(a) Hyperpolarized ¹³C-spectrum from an HCT116 Bax-ko cell assay showing the sum over the entire dynamic time series from a control cell experiment, following 6 hr starvation, 24 hr starvation with HBSS media and after 24 hr starvation followed by 48 hr cell recovery. The spectrum displays peaks from pyruvate (Pyr), lactate (Lac) and pyruvate hydrate (Pyr H). (b) Plot of the lactate peak integral as a function of time normalized to the pyruvate peak integral at time t = 0 s and normalized per million cells acquired for HCT116 Bax-ko control cells, 24 hr starvation with HBSS media and 24 hr starvation followed by 48 hr recovery. (c) The mean ratios (treated/24 hr vehicle-control) of the apparent forward reaction rate of pyruvate to lactate exchange k_PL (derived from fitting of the experimental data and normalized to cell number) (± s.e.m.) for 24 hr DMSO-treated recovery, 100 μM PI-103 treated and 48 hr recovery following 100 μM PI-103 treatment in HT29 cells; for 10 μM PI-103 treated, 20 μM PI-103 treated and 48 hr recovery following 20 μM PI-103 treatment in HCT116 Bax-ko cells; and for 6 hr HBSS starvation, 24 hr HBSS starvation and 24 hr starvation followed by 48 hr recovery in HCT116 Bax-ko cells. Minimum <i>n</i> = 3 in each group. Statistically significant changes are indicated (*p≤0.05).</p

Electron microscopy showing autophagic vacuoles.

<p>Electron microscopy images of autophagic vacuoles in control, 10 μM or 20 μM PI-103-treated or 6 hr and 24 hr HBSS-treated HCT116 Bax-ko cells. Red arrows illustrate some of the autophagic vacuoles at different stages of the autophagy process.</p

LDH activity and MCT-1 and MCT-4 expressions in PI-103- and HBSS-treated and its recovery in HT29 and HCT116 Bax-ko cells.

<p>(a) LDH activity ratios (treated/control) in PI-103-treated or HBSS-treated HT29 and HCT116 Bax-ko cells and levels at 48 hours recovery from 24 hr of PI-103-treatment or starvation. Minimum <i>n</i> = 3 in each group. Statistically significant changes are indicated (*p≤0.05). ⁺p = 0.06. (b) MCT-1 and MCT-4 expressions in PI-103-treated or HBSS-treated HT29 and HCT116 Bax-ko cells and levels at 48 hours recovery from 24 hr of PI-103-treatment or starvation.</p