RNF4 regulates DNA double-strand break repair in a cell cycle-dependent manner

<p>Both RNF4 and KAP1 play critical roles in the response to DNA double-strand breaks (DSBs), but the functional interplay of RNF4 and KAP1 in regulating DNA damage response remains unclear. We have previously demonstrated the recruitment and degradation of KAP1 by RNF4 require the phosphorylation of Ser824 (pS824) and SUMOylation of KAP1. In this report, we show the retention of DSB-induced pS824-KAP1 foci and RNF4 abundance are inversely correlated as cell cycle progresses. Following irradiation, pS824-KAP1 foci predominantly appear in the cyclin A (-) cells, whereas RNF4 level is suppressed in the G0-/G1-phases and then accumulates during S-/G2-phases. Notably, 53BP1 foci, but not BRCA1 foci, co-exist with pS824-KAP1 foci. Depletion of KAP1 yields opposite effect on the dynamics of 53BP1 and BRCA1 loading, favoring homologous recombination repair. In addition, we identify p97 is present in the RNF4-KAP1 interacting complex and the inhibition of p97 renders MCF7 breast cancer cells relatively more sensitive to DNA damage. Collectively, these findings suggest that combined effect of dynamic recruitment of RNF4 to KAP1 regulates the relative occupancy of 53BP1 and BRCA1 at DSB sites to direct DSB repair in a cell cycle-dependent manner.</p

Intermediary Metabolite Precursor Dimethyl-2-Ketoglutarate Stabilizes Hypoxia-Inducible Factor-1α by Inhibiting Prolyl-4-Hydroxylase PHD2

<div><p>Hypoxia-inducible factor 1α (HIF-1α), a major mediator of tumor physiology, is activated during tumor progression, and its abundance is correlated with therapeutic resistance in a broad range of solid tumors. The accumulation of HIF-1α is mainly caused by hypoxia or through the mutated succinate dehydrogenase A (SDHA) or fumarate hydratase (FH) expression to inhibit its degradation. However, its activation under normoxic conditions, termed pseudohypoxia, in cells without mutated SDHA or FH is not well documented. Here, we show that dimethyl-2-ketoglutarate (DKG), a cell membrane-permeable precursor of a key metabolic intermediate, α-ketoglutarate (α-KG), known for its ability to rescue glutamine deficiency, transiently stabilized HIF-1α by inhibiting activity of the HIF prolyl hydroxylase domain-containing protein, PHD2. Consequently, prolonged DKG-treatment under normoxia elevated HIF-1α abundance and up-regulated the expression of its downstream target genes, thereby inducing a pseudohypoxic condition. This HIF-1α stabilization phenotype is similar to that from treatment of cells with desferrioxamine (DFO), an iron chelator, or dimethyloxalyglycine (DMOG), an established PHD inhibitor, but was not recapitulated with other α-KG analogues, such as Octyl-2KG, MPTOM001 and MPTOM002. Our study is the first example of an α-KG precursor to increase HIF-1α abundance and activity. We propose that DKG acts as a potent HIF-1α activator, highlighting the potential use of DKG to investigate the contribution of PHD2-HIF-1α pathway to tumor biology.</p></div

DKG stabilizes HIF-1α by inhibiting HIF-1α proline hydroxylation and degradation.

<p>(<b>A</b>) DKG increases HIF-1α protein stability. MDA-MB-231 cells were pretreated with DKG (5 mM, 2-hour), then treated with CHX (20 µg/ml) in the presence or absence of DKG (5 mM) for an additional time period as indicated (<i>upper panel</i>). The relative abundance of HIF-1α is shown (<i>lower panel</i>). _*: <i>p</i><0.05; n = 3. (<b>B</b>) DKG blocks HIF-1α degradation mediated by PHD2. MDA-MB-231 cells were transfected with control siRNA or siRNAs targeting PHD1, PHD2 or PHD3 before treating with DKG (5 mM, 2-hour) (upper panel). <i>Italic numbers</i> represent the relative quantitation of protein levels. The level in the untreated sample from each pair of siCON-transfected, untreated cells was set as 1. The extent of respective knockdown of PHD1, 2 or 3 was assessed with semi-quantitative nested RT-PCR, followed by agarose gel electrophoresis (<i>lower panel</i>). (<b>C</b>) DKG increases HIF-1α stability through its oxygen-dependent degradation domain (ODD). ODD-luciferase activity was assayed in MDA-MB-231 cells co-transfected with ODD-luciferase reporter and control renilla luciferase reporter and treated with increasing doses of DKG for 6-hour. DMOG-treated cells serve a positive control. _*: <i>p</i><0.05; n = 3. (<b>D</b>) Hydroxylation at Pro564 of HIF-1α is inhibited by DKG. MDA-MB-231 cells were treated with the proteasome inhibitor, MG132, combined with the indicated chemicals to assess the level of HIF-1α hydroxylated at Pro564. The high-molecular weight, smeared species are the ubiquitylated HIF-1α. (<b>A</b>, <b>B</b>, <b>D</b>) A representative Western image from 3 independent experiments is shown.</p

DKG blocks HIF-1α degradation, rather than inducing its transcription or translation.

<p>(<b>A, B</b>) DKG increases HIF-1α abundance in both normoxic and hypoxic conditions. MDA-MB-231 (A) and MCF7 (B) cells were cultured in normoxia (21% O₂) or hypoxia (1% O₂) for 2-, 6- or 24-hour in the presence or absence of DKG (5 mM). (<b>C</b>) DKG reduces mTOR signaling. MDA-MB-231 cells were treated with DKG in the presence or absence of Gln. The signals of pT389-p70S6K and pT37/46-4EBP1 were used to as surrogate markers for mTOR activation. (<b>D</b>) DKG fails to further elevate HIF-1α protein abundance in cells pre-treated with MG132. MDA-MB-231 cells were pre-treated with a proteasome inhibitor, MG132 (5 µM, 4-hour) to block the proteasomal degradation. The cells were then continuously treated with MG132 and vehicle or DKG to assess HIF-1α abundance. (<b>E</b>) DKG, like DMOG or DFO, converges on proteasome-mediated degradation of HIF-1α. MDA-MB-231 cells treated with MG132 were co-treated with DKG (5 mM), DMOG (0.1 mM) or DFO (100 µM) for 6-hour and assessed for HIF-1α abundance. (<b>F</b>) Inhibiting PHD does not further increase the level of HIF-1α protein induced by DKG. MDA-MB-231 cells were treated with DKG (5 mM), DMOG (0.1 mM) or both for the indicated time periods under normoxia. (<b>E</b>–<b>F</b>) Numbers in <i>italic</i> represent the relative levels of HIF-1α protein. The level in untreated cells was set to 1. (<b>A</b>, <b>C</b>–<b>F</b>) A representative Western image from 3 independent experiments is shown. <i>Italic numbers</i> indicate the relative protein level after normalization with the level in the untreated cells set as 1.</p

DKG activates HIF-1α downstream signaling.

<p>(<b>A, B, C</b>) DKG induces HIF-1α target gene expression. <i>GLUT1 (A)</i>, <i>PDK1 (B) and CAIX (C)</i> mRNA abundance was assessed in MDA-MB-231/shCON and/shHIF-1α cells treated with DKG (5 mM, 6- or 16-hour) by quantitative PCR. _*: <i>p</i><0.05; _**: <i>p</i><0.01 (DKG-treated versus. untreated); #: <i>p</i><0.05; ##: <i>p</i><0.01 (shHIF-1α versus. shCON); n = 3. (<b>D</b>) CAIX is induced by DKG in a HIF-1α-dpenendent manner. CAIX protein level in the MDA-MB-231/shCON and/shHIF-1α cells treated with DKG (5 mM, 6- or 24-hour) was examined by Western blot analyses. A representative Western image from 3 independent experiments is shown. (<b>E</b>) DKG mediates a HIF-1α-dependent increase of <i>p21</i> mRNA abundance. <i>p21</i> mRNA abundance was measured in MDA-MB-231/shCON and/shHIF-1α cells treated with DKG (10 mM, 48-hour) by quantitative PCR. (<b>F</b>) HIF-1α activates <i>p21-Luc</i> reporter. <i>p21-Luc</i> reporter activity was quantitated by luciferase assays in HEK293 cells co-transfected with a combination of HIF-1α expression construct, <i>p21-Luc</i>, and renilla control reporter. (<b>G</b>) DKG inhibits cell proliferation. Cell proliferation was determined by measuring the activity of acid phosphatase (ACP) in the MDA-MB-231 cells treated with DKG (10 mM, 72-hour). (<b>H</b>) DKG does not induce apoptosis/cell death. Annexin V/PI double staining was performed to quantitate apoptosis/cell death in MDA-MB-231 cells treated with DKG (10 mM, 24-hour). (<b>I</b>) DKG pre-treatment renders doxorubicin resistance. MDA-MB-231 cells were pre-treated with DKG (10 mM, 7-day) and then treated with increasing doses of doxorubicin (0–400 nM). Cell proliferation was measured by ACP assay. _*: <i>p</i><0.05; _**: <i>p</i><0.01; n = 3.</p

(−)-Liriopein B Suppresses Breast Cancer Progression via Inhibition of Multiple Kinases

Numerous breast cancer patients who achieve an initial response to HER-targeted therapy rapidly develop resistance within one year, leading to treatment failure. Observations from clinical samples indicate that such resistance correlates with an increase in Src, EGFR, and PI3K/Akt activities and a decrease in PTEN activity. Furthermore, Akt survival signaling activation is also found in tumors treated by toxic chemotherapeutic agents. Because cotreatment with a PI3K inhibitor is a promising strategy to delay acquired resistance by preventing secondary gene activation, we therefore investigated the effects of a newly identified compound, (−)-Liriopein B (LB), on PI3K/Akt signaling activity in breast cancer cells. Our results showed that nontoxic doses of LB are able to inhibit AKT activation in both luminal-like MCF-7 and basal-like MDA-MB-231 breast cancer cells. Low doses of LB also inhibited cell migration, invasion, and cancer-stem cell sphere formation. Suppression of EGF-induced EGFR and ERK1/2 activation by LB might contribute in part to retardation of cancer progression. Furthermore, LB increases sensitivity of MDA-MB-231 cells to gefitinib in vitro, suggesting that EGFR may not be the only target of LB. Finally, a small scale in vitro kinase assay screen demonstrated that LB has a potent inhibitory effect on multiple kinases, including PI3K, Src, EGFR, Tie2, lck, lyn, RTK5, FGFR1, Abl, and Flt. In conclusion, this study demonstrates for the first time that the compound LB improves tumor therapeutic efficacy and suggests LB as a promising candidate for studying new leads in the development of kinase inhibitors

DKG promotes the HIF-1α accumulation in multiple cell lines under normoxia.

<p>(<b>A</b>) DKG elevates the steady-state HIF-1α abundance in non-cancerous cells. Human NHDF fibroblasts, stromal HS-5 cells and mammary epithelial MCF-10A cells were incubated with DKG under normoxia. The treatment with DFO (100 µM, 6-hour) to mimic hypoxia serves as a positive control. (<b>B</b>) DKG, but not Octyl-2KG, increases HIF-1α abundance in MDA-MB-231 cells under normoxia. MDA-MB-231 cells were treated with increasing doses of either DKG or Octyl-2KG for 2-hour. (<b>C</b>) Octyl-2KG decreases the steady-state HIF-1α protein abundance in a time-dependent manner. MDA-MB-231 cells were treated with Octyl-2KG (1 mM) for the indicated time periods. Cells treated with DKG (10 mM, 120-min) served as a positive control. (<b>D</b>) Octyl-2KG decreases HIF-1α protein level induced by DFO. MDA-MB-231 cells were treated with DFO (100 µM) and increasing doses of Octyl-2KG for 6-hour. (<b>E–F</b>) Octyl-2KG derivatives, MPTOM001 and MPTOM002, do not affect HIF-1α protein levels. MDA-MB-231 cells were treated with MPTOM001 or MPTOM002 in the absence (<b>E</b>) or presence (<b>F</b>) of DFO (100 µM). The chemical structures of DKG, Octyl-2KG, MPTOM001 and MPTOM002 are shown in (<b>C</b>, <b>D</b>, <b>E</b>). (<b>A–F</b>) Equal amount of whole cell lysates was analyzed by Western blots. GAPDH or Actin serves as a loading control. n = 3. A representative Western image from 3 independent experiments is shown. <i>Italic numbers</i> indicate the relative protein level after normalization with the level in the untreated cells.</p

(−)-Liriopein B Suppresses Breast Cancer Progression via Inhibition of Multiple Kinases

Numerous breast cancer patients who achieve an initial response to HER-targeted therapy rapidly develop resistance within one year, leading to treatment failure. Observations from clinical samples indicate that such resistance correlates with an increase in Src, EGFR, and PI3K/Akt activities and a decrease in PTEN activity. Furthermore, Akt survival signaling activation is also found in tumors treated by toxic chemotherapeutic agents. Because cotreatment with a PI3K inhibitor is a promising strategy to delay acquired resistance by preventing secondary gene activation, we therefore investigated the effects of a newly identified compound, (−)-Liriopein B (LB), on PI3K/Akt signaling activity in breast cancer cells. Our results showed that nontoxic doses of LB are able to inhibit AKT activation in both luminal-like MCF-7 and basal-like MDA-MB-231 breast cancer cells. Low doses of LB also inhibited cell migration, invasion, and cancer-stem cell sphere formation. Suppression of EGF-induced EGFR and ERK1/2 activation by LB might contribute in part to retardation of cancer progression. Furthermore, LB increases sensitivity of MDA-MB-231 cells to gefitinib in vitro, suggesting that EGFR may not be the only target of LB. Finally, a small scale in vitro kinase assay screen demonstrated that LB has a potent inhibitory effect on multiple kinases, including PI3K, Src, EGFR, Tie2, lck, lyn, RTK5, FGFR1, Abl, and Flt. In conclusion, this study demonstrates for the first time that the compound LB improves tumor therapeutic efficacy and suggests LB as a promising candidate for studying new leads in the development of kinase inhibitors

Autophagic reliance promotes metabolic reprogramming in oncogenic KRAS-driven tumorigenesis

<p>Defects in basal autophagy limit the nutrient supply from recycling of intracellular constituents. Despite our understanding of the prosurvival role of macroautophagy/autophagy, how nutrient deprivation, caused by compromised autophagy, affects oncogenic KRAS-driven tumor progression is poorly understood. Here, we demonstrate that conditional impairment of the autophagy gene <i>Atg5</i> (<i>atg5-</i>KO) extends the survival of KRAS^G12V-driven tumor-bearing mice by 38%. <i>atg5-</i>KO tumors spread more slowly during late tumorigenesis, despite a faster onset. <i>atg5-</i>KO tumor cells displayed reduced mitochondrial function and increased mitochondrial fragmentation. Metabolite profiles indicated a deficiency in the nonessential amino acid asparagine despite a compensatory overexpression of ASNS (asparagine synthetase), key enzyme for <i>de novo</i> asparagine synthesis. Inhibition of either autophagy or ASNS reduced KRAS^G12V-driven tumor cell proliferation, migration, and invasion, which was rescued by asparagine supplementation or knockdown of MFF (mitochondrial fission factor). Finally, these observations were reflected in human cancer-derived data, linking ASNS overexpression with poor clinical outcome in multiple cancers. Together, our data document a widespread yet specific asparagine homeostasis control by autophagy and ASNS, highlighting the previously unrecognized role of autophagy in suppressing the metabolic barriers of low asparagine and excessive mitochondrial fragmentation to permit malignant KRAS-driven tumor progression.</p