The epigenetic modifier HDAC2 and the checkpoint kinase ATM determine the responses of microsatellite instable colorectal cancer cells to 5-fluorouracil.

The epigenetic modifier histone deacetylase-2 (HDAC2) is frequently dysregulated in colon cancer cells. Microsatellite instability (MSI), an unfaithful replication of DNA at nucleotide repeats, occurs in about 15% of human colon tumors. MSI promotes a genetic frameshift and consequently a loss of HDAC2 in up to 43% of these tumors. We show that long-term and short-term cultures of colorectal cancers with MSI contain subpopulations of cells lacking HDAC2. These can be isolated as single cell-derived, proliferating populations. Xenografted patient-derived colon cancer tissues with MSI also show variable patterns of HDAC2 expression in mice. HDAC2-positive and HDAC2-negative RKO cells respond similarly to pharmacological inhibitors of the class I HDACs HDAC1/HDAC2/HDAC3. In contrast to this similarity, HDAC2-negative and HDAC2-positive RKO cells undergo differential cell cycle arrest and apoptosis induction in response to the frequently used chemotherapeutic 5-fluorouracil, which becomes incorporated into and damages RNA and DNA. 5-fluorouracil causes an enrichment of HDAC2-negative RKO cells in vitro and in a subset of primary colorectal tumors in mice. 5-fluorouracil induces the phosphorylation of KAP1, a target of the checkpoint kinase ataxia-telangiectasia mutated (ATM), stronger in HDAC2-negative cells than in their HDAC2-positive counterparts. Pharmacological inhibition of ATM sensitizes RKO cells to cytotoxic effects of 5-fluorouracil. These findings demonstrate that HDAC2 and ATM modulate the responses of colorectal cancer cells towards 5-FU.

Effect of particle size on the biodistribution, toxicity, and efficacy of drug-loaded polymeric nanoparticles in chemoradiotherapy.

Nanoparticle (NP) chemotherapeutics can improve the therapeutic index of chemoradiotherapy (CRT). However, the effect of NP physical properties, such particle size, on CRT is unknown. To address this, we examined the effects of NP size on biodistribution, efficacy and toxicity in CRT. PEG-PLGA NPs (50, 100, 150 nm mean diameters) encapsulating wotrmannin (wtmn) or KU50019 were formulated. These NP formulations were potent radiosensitizers in vitro in HT29, SW480, and lovo rectal cancer lines. In vivo, the smallest particles avoided hepatic and splenic accumulation while more homogeneously penetrating tumor xenografts than larger particles. However, smaller particles were no more effective in vivo. Instead, there was a trend toward enhanced efficacy with medium sized NPs. The smallest KU60019 particles caused more small bowel toxicity than larger particles. Our results showed that particle size significantly affects nanotherapeutics' biodistrubtion and toxicity but does not support the conclusion that smaller particles are better for this clinical application.

Inhibition of triple­negative breast cancer proliferation and motility by reactivating p53 and inhibiting overactivated Akt.

Mutations of p53 tumor suppressors occur more frequently in cancers at advanced stages or in more malignant cancer subtypes such as triple­negative breast cancer. Thus, restoration of p53 tumor suppressor function constitutes a valuable cancer therapeutic strategy. In the present study, it was revealed that a specific inhibitor of histone deacetylase 6, ACY­1215, caused increased acetylation of p53 in breast cancer cells with mutated p53, which was accompanied by increased expression of p21. These results suggested that ACY­1215 may lead to enhanced transcriptional activity of p53. It was also determined that ACY­1215 treatment resulted in G1 cell cycle arrest and apoptosis in these cancer cells. Furthermore, ACY­1215 displayed a synergistic effect with specific inhibitors of ATM, an activator of Akt, in inducing cancer cell apoptosis and inhibiting their motility. More importantly, it was observed that combination of ACY­1215 and ATM inhibitors exhibited markedly more potent antitumor activity than the individual compound in xenograft mouse models of breast cancer with mutant p53. Collectively, our results demonstrated that ACY­1215 is a novel chemotherapeutic agent that could restore mutant p53 function in cancer cells with strong antitumor activity, either alone or in combination with inhibitors of the ATM protein kinase.

Alleviation of Senescence via ATM Inhibition in Accelerated Aging Models.

The maintenance of mitochondrial function is closely linked to the control of senescence. In our previous study, we uncovered a novel mechanism in which senescence amelioration in normal aging cells is mediated by the recovered mitochondrial function upon Ataxia telangiectasia mutated (ATM) inhibition. However, it remains elusive whether this mechanism is also applicable to senescence amelioration in accelerated aging cells. In this study, we examined the role of ATM inhibition on mitochondrial function in Hutchinson-Gilford progeria syndrome (HGPS) and Werner syndrome (WS) cells. We found that ATM inhibition induced mitochondrial functional recovery accompanied by metabolic reprogramming, which has been known to be a prerequisite for senescence alleviation in normal aging cells. Indeed, the induced mitochondrial metabolic reprogramming was coupled with senescence amelioration in accelerated aging cells. Furthermore, the therapeutic effect via ATM inhibition was observed in HGPS as evidenced by reduced progerin accumulation with concomitant decrease of abnormal nuclear morphology. Taken together, our data indicate that the mitochondrial functional recovery by ATM inhibition might represent a promising strategy to ameliorate the accelerated aging phenotypes and to treat age-related disease.

Phosphatidylinositol 3 kinase (PI3K) modulates manganese homeostasis and manganese-induced cell signaling in a murine striatal cell line.

In a recent study, we found that blocking the protein kinase ataxia telangiectasia mutated (ATM) with the small molecule inhibitor (SMI) KU-55933 can completely abrogate Mn-induced phosphorylation of p53 at serine 15 (p-p53) in human induced pluripotent stem cell (hiPSC)-differentiated striatal neuroprogenitors. However, in the immortalized mouse striatal progenitor cell line STHdhQ7/Q7, a concentration of KU55933 far exceeding its IC50 for ATM was required to inhibit Mn-induced p-p53. This suggested an alternative signaling system redundant with ATM kinase for activating p53 in this cell line- one that was altered by KU55933 at these higher concentrations (i.e. mTORC1, DNApk, PI3K). To test the hypothesis that one or more of these signaling pathways contributed to Mn-induced p-p53, we utilized a set of SMIs (e.g. NU7441 and LY294002) known to block DNApk, PI3K, and mTORC1 at distinct concentrations. We found that the SMIs inhibit Mn-induced p-p53 expression near the expected IC50s for PI3K, versus other known targets. We hypothesized that inhibiting PI3K reduces intracellular Mn and thereby decreases activation of p53 by Mn. Using the cellular fura-2 manganese extraction assay (CFMEA), we determined that KU55933/60019, NU7441, and LY294002 (at concentrations near their IC50s for PI3K) all decrease intracellular Mn (∼50%) after a dual, 24-h Mn and SMI exposure. Many pathways are activated by Mn aside from p-p53, including AKT and mTOR pathways. Thus, we explored the activation of these pathways by Mn in STHdh cells as well as the effects of other pathway inhibitors. p-AKT and p-S6 activation by Mn is almost completely blocked upon addition of NU7441(5µM) or LY294002(7µM), supporting PI3K's upstream role in the AKT/mTOR pathway. We also investigated whether PI3K inhibition blocks Mn uptake in other cell lines. LY294002 exposure did not reduce Mn uptake in ST14A, Neuro2A, HEK293, MEF, or hiPSC-derived neuroprogenitors. Next, we sought to determine whether inhibition of PI3K blocked p53 phosphorylation by directly blocking an unknown PI3K/p53 interaction or indirectly reducing intracellular Mn, decreasing p-p53 expression. In-Cell Western and CFMEA experiments using multiple concentrations of Mn exposures demonstrated that intracellular Mn levels directly correlated with p-p53 expression with or without addition of LY294002. Finally, we examined whether PI3K inhibition was able to block Mn-induced p-p53 activity in hiPSC-derived striatal neuroprogenitors. As expected, LY294002 does not block Mn-induced p-p53 as PI3K inhibition is unable to reduce Mn net uptake in this cell line, suggesting the effect of LY294002 on Mn uptake is relatively specific to the STHdh mouse striatal cell line.

A crucial role for ATR in the regulation of deoxycytidine kinase activity.

Deoxycytidine kinase (dCK) (EC 2.7.1.74) is a key enzyme for salvage of deoxynucleosides and activation of numerous anticancer and antiviral nucleoside analogs. dCK activity is enhanced in response to several genotoxic treatments, which has been correlated with an increase of dCK phosphorylation at Ser-74. ATM was recently identified as the kinase responsible for Ser-74 phosphorylation and dCK activation after ionizing radiation (IR). Here, we investigated the role of ATM and the related kinase ATR in dCK activation induced by other types of DNA damage. Using ATM-deficient cells or the ATM inhibitor KU-60019, we found that ATM was not required for dCK activation caused by UV light, aphidicolin, cladribine, and unexpectedly also IR. On the other hand, the selective ATR inhibitor VE-821 significantly reduced up-regulation of dCK activity induced by these genotoxic agents, though not IR, and also down-regulated basal dCK activity. A role for ATR in the control of dCK activity was confirmed by using ATR siRNA and ATR-Seckel cells. ATR was also found to directly phosphorylate dCK at Ser-74 in vitro. Further studies revealed that ATR, which is also activated in response to IR, although later than ATM, was responsible for IR-induced dCK activation in ATM-deficient cells or in the presence of KU-60019. Overall, our results demonstrate that ATR controls basal dCK activity and dCK activation in response to replication stress and indicate that ATR can activate dCK after IR if ATM is lacking or inhibited.

Improved ataxia telangiectasia mutated kinase inhibitor KU60019 provides a promising treatment strategy for non-invasive breast cancer.

It has previously been reported that KU60019, as a highly effective radiosensitizer, inhibits the DNA damage response and blocks radiation-induced phosphorylation of key ataxia telangiectasia mutated targets in human glioma cells. The present study investigated whether KU60019 affects cell physiological activities and strengthens the efficacy of doxorubicin-induced DNA damage. It was demonstrated that the compound suppressed the proliferation of MCF-7 cells and significantly increased chemosensitization. In addition, KU60019 (without doxorubicin) inhibited MCF-7 cell motility and invasion, potentially by acting on the phosphorylated-Akt and E-cadherin signaling pathways. Although the majority of MCF-7 cells were arrested at the G1/S phase following treatment with KU60019, the combination of the two compounds did not result in such a marked effect on the cell cycle. In conclusion, KU60019 is a potent chemosensitizer in combination with doxorubicin, therefore, it may provide a promising strategy for non-invasive breast cancer.