Downregulation of AKT phosphorylation is necessary for induction of dormant status in primary colorectal cancer.

<p>A) CTOS growth was measured by size relative to day 0. C45 CTOS samples were cultured in medium with (GF+) or without (GF−) growth factors. B) Representative images of C45 CTOS cultured in indicated conditions. Scale bar  = 100 µm. C) Regrowth of CTOS in dormant status after re-oxygenation and exposure to growth factor–containing medium. D) Immunohistochemistry of C45 CTOS cultured in indicated conditions for 1 day. TUNEL staining was at day 14. Scale bar  = 50 µm. E) Immunoblot of AKT/mTORC1 signaling and HIF-1α in C45 CTOS cultured in indicated conditions.</p

The role of apoptosis in hypoxia-induced gene downregulation in MIN6 cells.

<p>(A) MIN6 cells were incubated at 5% O2 for 10 h and the expression levels of <i>Ddit3</i> mRNA were examined by qPCR (n = 4). (B, C) Annexin V-positive cell (B) and PI-positive cell death (C) ratios were evaluated by flow cytometric analysis (n = 6) after MIN6 cells were cultured at 5% O2 for 10 h. (D) Expression levels of <i>Pdx1</i>, <i>Neurod1</i>, <i>Wfs1</i>, and <i>Slc2a2</i> were examined by qPCR (n = 4) in the same conditions as in (A). (E) Ctrl MIN6 cells (gray bars) and CHOP knockdown MIN6 cells (black bars) were incubated either in normoxia (20% O₂) or in hypoxia (5% O₂) for 40 h and the expression levels of <i>Ddit3</i> mRNA were examined by qPCR (n = 3). (F) Ctrl MIN6 cells (gray bars) (n = 5) and CHOP knockdown MIN6 cells (black bars) (n = 5) were incubated either in normoxia (20% O₂) or in hypoxia (5% O₂). The PI-positive cell death ratio was evaluated by flow cytometric analysis. (G) Ctrl MIN6 cells (gray bars) and CHOP knockdown MIN6 cells (black bars) were incubated in the same conditions as in (E). Downregulation of mRNA levels (Δ mRNA levels) by 5% O₂. Δ mRNA levels indicate (1-expression levels at 5% O₂/expression levels at 20% O₂) ×100 (%). In qPCR analysis, the mRNA value of each gene was normalized to that of <i>Tbp</i>. The data are shown as the means ± S.E. (error bars) of values from each group. *, p<0.05; **, p<0.01; ***, p<0.001.</p

Downregulation of AKT phosphorylation is important for induction of dormant status.

<p>A) Immunoblot of the AKT/mTORC1 or ERK/p38 MAPK pathway in AsPC-1 cells cultured in hypoxia. B) Immunoblot of AKT signaling and HIF-1α in AsPC-1 cells expressing vector control, AKT-wt, or AKT-3A (inactive). C) Cell cycle status of the cells at day 7 in hypoxia. Percentages of the cells in S phase are indicated above the plot and in the right graph. D) ATP turnover measured by adding inhibitor cocktail for glycolysis and oxidative phosphorylation. N1, normoxia 1 day; H1, hypoxia 1 day; H7, hypoxia 7 days. E, F) Viable cell number (E) and percent cell death (F) of AsPC-1 cells cultured in hypoxia. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

AsPC-1 cells can be in a dormant status in chronic hypoxia.

<p>Viable cell number (A) and percent cell death (B) of AsPC-1 cells cultured in normoxia (20% O₂) or hypoxia (1% O₂). C) Phase-contrast and PI-stained images of AsPC-1 cells cultured under the indicated conditions. Scale bar  = 50 µm. D) Cell cycle analysis of AsPC-1 cells at day 1 in normoxia or day 1 and day 7 in hypoxia. Cells were pulsed with BrdU for 2 h and analyzed by flow cytometry after staining with anti-BrdU antibody and PI. Percentages of the cells in S phase are indicated. E) Re-growth of AsPC-1 cells in a dormant status. AsPC-1 cells were cultured in hypoxia for 14 days, and the cell counts were monitored after re-seeding in normoxic conditions.</p

Dormancy of Cancer Cells with Suppression of AKT Activity Contributes to Survival in Chronic Hypoxia

<div><p>A hypoxic microenvironment in tumors has been recognized as a cause of malignancy or resistance to various cancer therapies. In contrast to recent progress in understanding the acute response of cancer cells to hypoxia, the characteristics of tumor cells in chronic hypoxia remain elusive. We have identified a pancreatic cancer cell line, AsPC-1, that is exceptionally able to survive for weeks under 1% oxygen conditions while most tested cancer cell lines die after only some days under these conditions. In chronic hypoxia, AsPC-1 cells entered a state of dormancy characterized by no proliferation, no death, and metabolic suppression. They reversibly switched to active status after being placed again in optimal culture conditions. ATP turnover, an indicator of energy demand, was markedly decreased and accompanied by reduced AKT phosphorylation. Forced activation of AKT resulted in increased ATP turnover and massive cell death <i>in vitro</i> and a decreased number of dormant cells <i>in vivo</i>. In contrast to most cancer cell lines, primary-cultured colorectal cancer cells easily entered the dormant status with AKT suppression under hypoxia combined with growth factor–depleted conditions. Primary colorectal cancer cells in dormancy were resistant to chemotherapy. Thus, the ability to survive in a deteriorated microenvironment by entering into dormancy under chronic hypoxia might be a common property among cancer cells. Targeting the regulatory mechanism inducing this dormant status could provide a new strategy for treating cancer.</p></div

Moderate Hypoxia Induces β-Cell Dysfunction with HIF-1–Independent Gene Expression Changes

<div><p>Pancreatic β-cell failure is central to the development and progression of type 2 diabetes. We recently demonstrated that β-cells become hypoxic under high glucose conditions due to increased oxygen consumption and that the pancreatic islets of diabetic mice but not those of control mice are moderately hypoxic. However, the impact of moderate hypoxia on β-cell number and function is unknown. In the present study, moderate hypoxia induced a hypoxic response in MIN6 cells, as evidenced by increased levels of HIF-1α protein and target genes. Under these conditions, a selective downregulation of <i>Mafa</i>, <i>Pdx1</i>, <i>Slc2a2</i>, <i>Ndufa5</i>, <i>Kcnj11</i>, <i>Ins1</i>, <i>Wfs1</i>, <i>Foxa2</i>, and <i>Neurod1</i>, which play important roles in β-cells, was also observed in both MIN6 cells and isolated pancreatic islets. Consistent with the altered expression of these genes, abnormal insulin secretion was detected in hypoxic MIN6 cells. Most of the hypoxia-induced gene downregulation in MIN6 cells was not affected by the suppression of HIF-1α, suggesting a HIF-1–independent mechanism. Moderate hypoxia also induced apoptosis in MIN6 cells. These results suggest that hypoxia is a novel stressor of β-cells and that hypoxic stress may play a role in the deterioration of β-cell function.</p></div

Metabolic processes are suppressed under chronic hypoxia.

<p>A) Lactate production rate (left) was calculated from lactate concentration and integral cell number at indicated periods. O₂ consumption rate (middle) was measured using a Clark type oxygen electrode. ATP turnover (right) was calculated from the lactate production rate and O₂ consumption rate (dark gray: lactate; light gray: oxygen). N1, normoxia 1 day; H1, hypoxia 1 day; H7, hypoxia 7 days. ** <i>p</i><0.01, ***<i>p</i><0.001. B) Quantitative RT-PCR of a glucose transporter and glycolytic enzymes in AsPC-1 cells cultured in hypoxia for the indicated days.</p

Altered insulin secretion by MIN6 cells under hypoxia.

<p>(A) Cellular insulin content was examined after MIN6 cells were incubated in normoxia (20% O₂, gray bars) or hypoxia (5% O₂, black bars) for 30 h. Insulin content was standardized by cell number. (B) Glucose-stimulated insulin secretion was examined when MIN6 cells that had been cultured at 20% O₂ or 5% O₂ for 40 h were stimulated with low glucose (2.2 mM glucose, gray bars) or high glucose (22 mM glucose, black bars) for 1 h (n = 4). Secreted insulin was normalized to cellular protein levels. Data are shown as the means ± S.E. (error bars) of values from each group. **, p<0.01; ***, p<0.001. N.S., not significant.</p

The role of HIF-1 in hypoxia-induced gene downregulation and insulin secretion defects in MIN6 cells.

<p>(A) Control (Ctrl) MIN6 cells (gray bars) and HIF-1α knockdown MIN6 cells (black bars) were cultured in normoxia (20% O₂) and the relative expression levels of β-cell genes were evaluated by qPCR analysis. Each value of mRNA was normalized to that of <i>Actb</i>. (B) Downregulation of mRNA levels (Δ mRNA levels) by 5% O₂. Δ mRNA levels indicate (1-expression levels at 5% O₂/expression levels at 20% O₂) ×100 (%). (C) Ctrl MIN6 cells (n = 11) and HIF-1α knockdown MIN6 cells (n = 9) that had been cultured in normoxic (20% O₂, gray bars) or hypoxic (5% O_2, black bars) conditions for 40 h were stimulated with 2.2 mM glucose or 22 mM glucose for 1 h. Secreted insulin was normalized to cellular protein levels. (D) The fold change in glucose-stimulated insulin secretion (insulin level at 22 mM glucose divided by that at 2.2 mM glucose) is indicated (n = 9). Data are shown as the means ± S.E. (error bars) of the values from each group. *, p<0.05; **, p<0.01; ***, p<0.001.</p

The effect of moderate hypoxia on β-cell gene expression.

<p>(A, B) Gene expression analysis by qPCR of β-cell transcription factors (A) and components of the insulin secretion pathway (B) was performed using MIN6 cells incubated in normoxia (20% O₂, gray bars; n = 4) or in hypoxia (5% O₂, black bars; n = 4) for 30 h. (C) Gene expression analysis by qPCR was performed when mouse isolated islets were cultured in normoxia (20% O₂, gray bars; n = 3) or hypoxia (5% O₂, black bars; n = 3) for 16 h. (D) MIN6 cells were incubated in normoxia (20% O₂, gray bars; n = 4) or in hypoxia (5% O₂, black bars; n = 4) for 16 h. The mRNA value of each gene was normalized to that of <i>Tbp</i>. The means ± S.E. (error bars) of values from each group are shown. *, p<0.05; **, p<0.01; ***, p<0.001.</p