Molecular pathways and targets in prostate cancer.

Prostate cancer co-opts a unique set of cellular pathways in its initiation and progression. The heterogeneity of prostate cancers is evident at earlier stages, and has led to rigorous efforts to stratify the localized prostate cancers, so that progression to advanced stages could be predicted based upon salient features of the early disease. The deregulated androgen receptor signaling is undeniably most important in the progression of the majority of prostate tumors. It is perhaps because of the primacy of the androgen receptor governed transcriptional program in prostate epithelium cells that once this program is corrupted, the consequences of the ensuing changes in activity are pleotropic and could contribute to malignancy in multiple ways. Following localized surgical and radiation therapies, 20-40% of patients will relapse and progress, and will be treated with androgen deprivation therapies. The successful development of the new agents that inhibit androgen signaling has changed the progression free survival in hormone resistant disease, but this has not changed the almost ubiquitous development of truly resistant phenotypes in advanced prostate cancer. This review summarizes the current understanding of the molecular pathways involved in localized and metastatic prostate cancer, with an emphasis on the clinical implications of the new knowledge.

Pathways and therapeutic targets in melanoma.

This review aims to summarize the current knowledge of molecular pathways and their clinical relevance in melanoma. Metastatic melanoma was a grim diagnosis, but in recent years tremendous advances have been made in treatments. Chemotherapy provided little benefit in these patients, but development of targeted and new immune approaches made radical changes in prognosis. This would not have happened without remarkable advances in understanding the biology of disease and tremendous progress in the genomic (and other "omics") scale analyses of tumors. The big problems facing the field are no longer focused exclusively on the development of new treatment modalities, though this is a very busy area of clinical research. The focus shifted now to understanding and overcoming resistance to targeted therapies, and understanding the underlying causes of the heterogeneous responses to immune therapy.

Molecular pathways and therapeutic targets in lung cancer.

Lung cancer is still the leading cause of cancer death worldwide. Both histologically and molecularly lung cancer is heterogeneous. This review summarizes the current knowledge of the pathways involved in the various types of lung cancer with an emphasis on the clinical implications of the increasing number of actionable molecular targets. It describes the major pathways and molecular alterations implicated in the development and progression of non-small cell lung cancer (adenocarcinoma and squamous cancer), and of small cell carcinoma, emphasizing the molecular alterations comprising the specific blueprints in each group. The approved and investigational targeted therapies as well as the immune therapies, and clinical trials exploring the variety of targeted approaches to treatment of lung cancer are the main focus of this review.

Bezielle selectively targets mitochondria of cancer cells to inhibit glycolysis and OXPHOS.

Bezielle (BZL101) is a candidate oral drug that has shown promising efficacy and excellent safety in the early phase clinical trials for advanced breast cancer. Bezielle is an aqueous extract from the herb Scutellaria barbata. We have reported previously that Bezielle was selectively cytotoxic to cancer cells while sparing non-transformed cells. In tumor, but not in non-transformed cells, Bezielle induced generation of ROS and severe DNA damage followed by hyperactivation of PARP, depletion of the cellular ATP and NAD, and inhibition of glycolysis. We show here that tumor cells' mitochondria are the primary source of reactive oxygen species induced by Bezielle. Treatment with Bezielle induces progressively higher levels of mitochondrial superoxide as well as peroxide-type ROS. Inhibition of mitochondrial respiration prevents generation of both types of ROS and protects cells from Bezielle-induced death. In addition to glycolysis, Bezielle inhibits oxidative phosphorylation in tumor cells and depletes mitochondrial reserve capacity depriving cells of the ability to produce ATP. Tumor cells lacking functional mitochondria maintain glycolytic activity in presence of Bezielle thus supporting the hypothesis that mitochondria are the primary target of Bezielle. The metabolic effects of Bezielle towards normal cells are not significant, in agreement with the low levels of oxidative damage that Bezielle inflicts on them. Bezielle is therefore a drug that selectively targets cancer cell mitochondria, and is distinguished from other such drugs by its ability to induce not only inhibition of OXPHOS but also of glycolysis. This study provides a better understanding of the mechanism of Bezielle's cytotoxicity, and the basis of its selectivity towards cancer cells.

Identification and analysis of the active phytochemicals from the anti-cancer botanical extract Bezielle.

Bezielle is a botanical extract that has selective anti-tumor activity, and has shown a promising efficacy in the early phases of clinical testing. Bezielle inhibits mitochondrial respiration and induces reactive oxygen species (ROS) in mitochondria of tumor cells but not in non-transformed cells. The generation of high ROS in tumor cells leads to heavy DNA damage and hyper-activation of PARP, followed by the inhibition of glycolysis. Bezielle therefore belongs to a group of drugs that target tumor cell mitochondria, but its cytotoxicity involves inhibition of both cellular energy producing pathways. We found that the cytotoxic activity of the Bezielle extract in vitro co-purified with a defined fraction containing multiple flavonoids. We have isolated several of these Bezielle flavonoids, and examined their possible roles in the selective anti-tumor cytotoxicity of Bezielle. Our results support the hypothesis that a major Scutellaria flavonoid, scutellarein, possesses many if not all of the biologically relevant properties of the total extract. Like Bezielle, scutellarein induced increasing levels of ROS of mitochondrial origin, progressive DNA damage, protein oxidation, depletion of reduced glutathione and ATP, and suppression of both OXPHOS and glycolysis. Like Bezielle, scutellarein was selectively cytotoxic towards cancer cells. Carthamidin, a flavonone found in Bezielle, also induced DNA damage and oxidative cell death. Two well known plant flavonoids, apigenin and luteolin, had limited and not selective cytotoxicity that did not depend on their pro-oxidant activities. We also provide evidence that the cytotoxicity of scutellarein was increased when other Bezielle flavonoids, not necessarily highly cytotoxic or selective on their own, were present. This indicates that the activity of total Bezielle extract might depend on a combination of several different compounds present within it.

Bezielle (BZL101)-induced oxidative stress damage followed by redistribution of metabolic fluxes in breast cancer cells: a combined proteomic and metabolomic study.

Bezielle is an orally administered aqueous extract of Scutellaria barbata for treatment of advanced and metastatic breast cancer. Phase I trials showed promising tolerability and efficacy. In our study, we used a combined proteomic-metabolomic approach to investigate the molecular pathways affected by Bezielle in ER-positive BT474 and ER-negative SKBR3 cell lines. In both, Bezielle inhibited cell proliferation, induced cell death and G2 cycle arrest by regulating the mediator proteins Jab1, p27(Kip1) and p21(Cip1) . In addition, it stimulated reactive oxygen species production, hyperactivation of PARP and inhibition of glycolysis. Bezielle's ability to induce oxidative stress was associated with the changes in expression of redox potential maintaining enzymes: glutathione- and thioredoxin-related proteins and peroxiredoxins. In regards to cell metabolism, decreased expression of α-enolase was associated with a reduction of de novo (13) C-lactate formation. Reduced Krebs cycle activity as evidenced by the reduced expression of α-ketoglutarate dehydrogenase and succinyl-CoA synthetase led to decreased intracellular succinate concentrations. By inhibiting glucose metabolism, cells reacted by lowering the expression of glucose transporters and resulting in decreased intracellular glucose concentration. Decreased expression of fatty acid synthase and reduced concentration of phosphocholine indicated considerable changes in phospholipid metabolism. Ultimately, by inhibiting the major energy-producing pathways, Bezielle caused depletion of ATP and NAD(H). Both cell lines were responsive, thus suggesting that Bezielle has the potential to be effective against ER-negative breast cancers. In conclusion, Bezielle's cytotoxicity toward cancer cells is primarily based on inhibition of metabolic pathways that are preferentially activated in tumor cells thus explaining its specificity for cancer cells.

Estrogen receptor ß causes a G2 cell cycle arrest by inhibiting CDK1 activity through the regulation of cyclin B1, GADD45A, and BTG2.

The role of estrogen receptor beta (ERß) in breast cancer is unclear. ERß is considered to have a protective role in breast cancer development based on findings demonstrating that ERß expression inhibits ERα-mediated proliferation of breast cancer cells. We previously demonstrated that ERß causes a ligand independent G2 cell cycle arrest in MCF-7 cells. To study the mechanisms of the ERß-mediated G2 cell cycle arrest, we investigated its effects on the regulatory pathways responsible for the G2/M phase transition. We found that ERß inhibits CDK1 activity, which is the critical determinant of the G2/M progression. CDK1 activity is modulated by both stimulatory and inhibitory factors. Cyclin B1 is the major activator of CDK1. ERß inhibited the cell cycle-dependent stimulation of cyclin B1 mRNA and protein. GADD45A and BTG2 are two major inhibitors of CDK1, which have been implicated in breast tumor formation. Based on these findings, we explored if the expression pattern of GADD45A and BTG2 is affected by ERß. We found that ERß stimulates GADD45A and BTG2 mRNA levels. The induction of these two genes is caused by ERß binding directly to these genes and recruiting c-jun and NCOA2. Our findings demonstrated that unliganded ERß causes a G2 cell cycle arrest by inactivating CDK1 through the repression of cyclin B1 and stimulation of GADD45A and BTG2 expression. These results provide evidence that drugs that stimulate the production of unliganded ERß may be effective new therapies to prevent breast cancer.

CC3/TIP30 regulates metabolic adaptation of tumor cells to glucose limitation.

CC3/TIP30 is a metastasis and tumor suppressor, with reduced or absent expression in a variety of aggressive tumors. Overexpression of CC3 in tumor cells predisposes them to apoptosis in response to different death signals. We found that silencing of CC3 expression does not increase apoptotic resistance of cells. However, it strongly improves survival of tumor cells in response to glucose limitation. HeLa cells with silenced CC3 survive long-term in low glucose, and, in comparison to control HeLa cells, show superior metabolic adaptation to glucose limitation. First, unlike the parental HeLa cells, HeLa with silenced CC3 activate and maintain high levels of mitochondrial respiration that is critical for their ability to thrive in low glucose. Second, silencing of CC3 leads to higher expression levels of mitochondrial proteins in respiration complexes when cells are continuously cultured in limiting glucose. Third, HeLa cells with silenced CC3 maintain higher levels of c-MYC and the M2 isoform of pyruvate kinase in low glucose, contributing to more efficient glycolysis. Fourth, HeLa cells with silenced CC3 fail to fully activate AMPK in response to glucose limitation. Inhibition of AMPK, either pharmacologic or via siRNA, protects control HeLa cells from death in low glucose. The metabolic flexibility acquired by cells after silencing of CC3 could be directly relevant to the development of metastatic and aggressive human tumors that frequently have low or absent expression of CC3.

Vascular endothelial platelet endothelial cell adhesion molecule 1 (PECAM-1) regulates advanced metastatic progression.

Most patients who die from cancer succumb to treatment-refractory advanced metastatic progression. Although the early stages of tumor metastasis result in the formation of clinically silent micrometastatic foci, its later stages primarily reflect the progressive, organ-destructive growth of already advanced metastases. Early-stage metastasis is regulated by multiple factors within tumor cells as well as by the tumor microenvironment (TME). In contrast, the molecular determinants that control advanced metastatic progression remain essentially uncharacterized, precluding the development of therapies targeted against it. Here we show that the TME, functioning in part through platelet endothelial cell adhesion molecule 1 (PECAM-1), drives advanced metastatic progression and is essential for progression through its preterminal end stage. PECAM-1-KO and chimeric mice revealed that its metastasis-promoting effects are mediated specifically through vascular endothelial cell (VEC) PECAM-1. Anti-PECAM-1 mAb therapy suppresses both end-stage metastatic progression and tumor-induced cachexia in tumor-bearing mice. It reduces proliferation, but not angiogenesis or apoptosis, within advanced tumor metastases. Because its antimetastatic effects are mediated by binding to VEC rather than to tumor cells, anti-PECAM-1 mAb appears to act independently of tumor type. A modified 3D coculture assay showed that anti-PECAM-1 mAb inhibits the proliferation of PECAM-1-negative tumor cells by altering the concentrations of secreted factors. Our studies indicate that a complex interplay between elements of the TME and advanced tumor metastases directs end-stage metastatic progression. They also suggest that some therapeutic interventions may target late-stage metastases specifically. mAb-based targeting of PECAM-1 represents a TME-targeted therapeutic approach that suppresses the end stages of metastatic progression, until now a refractory clinical entity.

CC3/TIP30 affects DNA damage repair.

BACKGROUND: The pro-apoptotic protein CC3/TIP30 has an unusual cellular function as an inhibitor of nucleocytoplasmic transport. This function is likely to be activated under conditions of stress. A number of studies support the notion that CC3 acts as a tumor and metastasis suppressor in various types of cancer. The yeast homolog of CC3 is likely to be involved in responses to DNA damage. Here we examined the potential role of CC3 in regulation of cellular responses to genotoxic stress. RESULTS: We found that forced expression of CC3 in CC3-negative cells strongly delays the repair of UV-induced DNA damage. Exogenously introduced CC3 negatively affects expression levels of DDB2/XPE and p21CIP1, and inhibits induction of c-FOS after UV exposure. In addition, exogenous CC3 prevents the nuclear accumulation of P21CIP in response to UV. These changes in the levels/localization of relevant proteins resulting from the enforced expression of CC3 are likely to contribute to the observed delay in DNA damage repair. Silencing of CC3 in CC3-positive cells has a modest delaying effect on repair of the UV induced damage, but has a much more significant negative affect on the translesion DNA synthesis after UV exposure. This could be related to the higher expression levels and increased nuclear localization of p21CIP1 in cells where expression of CC3 is silenced. Expression of CC3 also inhibits repair of oxidative DNA damage and leads to a decrease in levels of nucleoredoxin, that could contribute to the reduced viability of CC3 expressing cells after oxidative insult. CONCLUSIONS: Manipulation of the cellular levels of CC3 alters expression levels and/or subcellular localization of proteins that exhibit nucleocytoplasmic shuttling. This results in altered responses to genotoxic stress and adversely affects DNA damage repair by affecting the recruitment of adequate amounts of required proteins to proper cellular compartments. Excess of cellular CC3 has a significant negative effect on DNA repair after UV and oxidant exposure, while silencing of endogenous CC3 slightly delays repair of UV-induced damage.

A phase 1B dose escalation trial of Scutellaria barbata (BZL101) for patients with metastatic breast cancer.

The purpose of this study was to determine the safety and maximum tolerated dose (MTD) of BZL101 (FDA IND# 59,521), an orally delivered aqueous extract from the herb Scutellaria barbata, in women with metastatic breast cancer (MBC). The trial was an open-label, phase 1B, multicenter, dose escalation study. Eligible patients had histologically confirmed breast cancer and measurable stage IV disease. The standard phase 1 "3 + 3" study design was used to determine the MTD. Primary endpoints were toxicity and MTD of BZL101. Secondary outcomes included efficacy based on RECIST criteria. A total of 27 women with a median of 2 prior chemotherapy treatments for metastatic disease were treated in four different dose cohorts. Grade 3 and 4 adverse events (AEs) were uncommon. Dose-limiting toxicities included the following: grade 4 AST elevation, grade 3 diarrhea, grade 3 fatigue, and grade 3 rib pain. Fourteen patients were evaluable according to Response Evaluation Criteria in Solid Tumors. Investigator assessment classified three patients with stable disease for >120 days (21%). One patient was on BZL101 for 449 days and remains stable for 700 + days. Independent radiology review identified three patients with objective tumor regression (>0% and <30%). The MTD was not reached, thus per protocol, the MTD was defined as the maximum administered dose of BZL101 40 g/day. In conclusion, oral administration of BZL101 was safe, well tolerated, and showed promising clinical evidence of anticancer activity in this heavily pretreated population of women with MBC.

Selective concomitant inhibition of mTORC1 and mTORC2 activity in estrogen receptor negative breast cancer cells by BN107 and oleanolic acid.

Hormonal, targeted and chemotherapeutic strategies largely depend on the expression of their cognate receptors and are often accompanied by intolerable toxicities. Effective and less toxic therapies for estrogen receptor negative (ER-) breast cancers are urgently needed. Here, we present the potential molecular mechanisms mediating the selective pro-apoptotic effect induced by BN107 and its principle terpene, oleanolic acid (OA), on ER- breast cancer cells. A panel of breast cancer cell lines was examined and the most significant cytotoxic effect was observed in ER- breast lines. Apoptosis was the major cellular pathway mediating the cytotoxicity of BN107. We demonstrated that sensitivity to BN107 was correlated to the status of ERalpha. Specifically, the presence of functional ERalpha protected cells from BN107-induced apoptosis and absence of ERalpha increased the sensitivity. BN107, an extract rich in OA derivatives, caused rapid alterations in cholesterol homeostasis, presumably by depleting cholesterol in lipid rafts (LRs), which subsequently interfered with signaling mediated by LRs. We showed that BN107 or OA treatment in ER- breast cancer cells resulted in rapid and specific inhibition of LR-mediated survival signaling, namely mTORC1 and mTORC2 activities, by decreasing the levels of the mTOR/FRAP1, RAPTOR and RICTOR. Cotreatment with cholesterol abolished the proapoptotic effect and restored the disrupted mTOR activities. This is the first report demonstrating possible concomitant inhibition of both mTORC1 and mTORC2 activities by modulating the levels of protein constituents present in these signaling complexes, and thus provides a basis for future development of OA-based mTOR inhibitors.

Timosaponin AIII is preferentially cytotoxic to tumor cells through inhibition of mTOR and induction of ER stress.

The aqueous extract of Anemarrhena asphodeloides (BN108) induces apoptosis in various cancer cell lines but is significantly less cytotoxic in non-transformed cells. Chemical fractionation of BN108 showed that its cytotoxicity is associated with timosaponins, steroidal saponins of coprostane type. Timosaponin BII (TBII) is a major saponin in BN108, but it shows little cytotoxicity. A much less abundant TAIII induces cell death in tumor cells but not in normal cells, reproducing the selectivity of the total extract BN108. Glycosidase treatment, by removing the extra sugar moiety in TBII, converts it to TAIII and confers cytotoxic activity. Analysis of the mechanisms of death induced by TAIII revealed activation of two distinct pro-apoptotic pathways: first, inhibition of mTORC1 manifested in much reduced phosphorylation of mTORC1 targets; second, induction of endoplasmic reticulum stress culminating in phosphorylation of eIF2alpha and activation of caspase 4. These pro-apoptotic pathways are activated by TAIII selectively in tumor cells but not in normal cells. Both pathways play a causative role in TAIII cytotoxicity, as restoration of either mTOR activity or relief of ER stress alone offer only partial protection from TAIII. Inhibition of mTORC1 and induction of ER stress apparently contribute to the induction of the previously reported autophagic response in TAIII-treated cells. TAIII induced autophagy plays a protective role in TAIII induced death signaling, and failure to mount autophagic response is associated with heightened sensitivity to TAIII induced apoptosis. The multiple death-promoting and apparently tumor-selective responses to TAIII, its ability to inhibit mTORC1, and the possibility of further enhancing its cytotoxicity by pharmacological inhibition of autophagy, make TAIII an attractive candidate for development as a cancer therapeutic agent.

Molecular mechanisms underlying selective cytotoxic activity of BZL101, an extract of Scutellaria barbata, towards breast cancer cells.

We studied the mechanism of the cytotoxic activity of BZL101, an aqueous extract from the herb Scutellaria barbata D. Don, which is currently in phase II clinical trial in patients with advanced breast cancer. The phase I trial showed favorable toxicity profile and promising efficacy. We report here that BZL101 induces cell death in breast cancer cells but not in non-transformed mammary epithelial cells. This selective cytotoxicity is based on strong induction by BZL101 of reactive oxygen species (ROS) in tumor cells. As a consequence, BZL101 treated cancer cells develop extensive oxidative DNA damage and succumb to necrotic death. Data from the expression profiling of cells treated with BZL101 are strongly supportive of a death pathway that involves oxidative stress, DNA damage and activation of death-promoting genes. In breast cancer cells oxidative damage induced by BZL101 leads to the hyperactivation of poly (ADP-ribose) polymerase (PARP), followed by a sustained decrease in levels of NAD and depletion of ATP, neither of which are observed in non-transformed cells. The hyperactivation of PARP is instrumental in the necrotic death program induced by BZL101, because inhibition of PARP results in suppression of necrosis and activation of the apoptotic death program. BZL101 treatment leads to the inhibition of glycolysis selectively in tumor cells, evident from the decrease in the enzymatic activities within the glycolytic pathway and the inhibition of lactate production. Because tumor cells frequently rely on glycolysis for energy production, the observed inhibition of glycolysis is likely a key factor in the energetic collapse and necrotic death that occurs selectively in breast cancer cells. The promising selectivity of BZL101 towards cancer cells is based on metabolic differences between highly glycolytic tumor cells and normal cells.

Phase I trial and antitumor effects of BZL101 for patients with advanced breast cancer.

BACKGROUND: Botanical therapies are often used by breast cancer patients yet few clinical trials have evaluated their safety and efficacy. We studied mechanisms of activity and performed a phase I clinical trial in patients with advanced breast cancer to evaluate BZL101, an aqueous extract from Scutellaria barbata. METHODS: Preclinical studies were conducted in vitro to characterize cell death induced by BZL101. In a phase I trial, eligible patients had histologically confirmed, measurable metastatic breast cancer. Treatment consisted of 350 ml per day of oral BZL101, administered as sole cancer therapy until disease progression, toxicity or personal preference to discontinue. Primary endpoints were safety, toxicity and tumor response. RESULTS: BZL101 extract induced strong growth inhibition and apoptosis of breast cancer cell lines. In the phase I trial, 21 patients received BZL101. Mean age was 54 years (30-77) and mean number of prior treatments for metastatic disease was 3.9 (0-10). There were no grade III or IV adverse events (AEs). The most frequently reported BZL101-related grade I and II AEs included: nausea (38%), diarrhea (24%), headache (19%) flatulence (14%), vomiting (10%), constipation (10%), and fatigue (10%). Sixteen patients were evaluable for response. Four patients had stable disease (SD) for >90 days (25%) and 3/16 had SD for >180 days (19%). Five patients had objective tumor regression, one of which was 1 mm short of a PR based on RECIST criteria. CONCLUSIONS: BZL 101 inhibits breast cancer cell lines by inducing apoptosis. In a phase I clinical trial, BZL101 was safe and had a favorable toxicity profile. BZL101 demonstrated encouraging clinical activity in this heavily pretreated population.

Inhibition of nuclear import by the proapoptotic protein CC3.

We report here that the normal cellular protein CC3/TIP30, when in excess, inhibits nuclear import in vitro and in vivo. CC3 binds directly to the karyopherins of the importin beta family in a RanGTP-insensitive manner and associates with nucleoporins in vivo. CC3 inhibits the nuclear import of proteins possessing either the classical nuclear localization signal or the M9 signal recognized by transportin. CC3 also inhibits nuclear translocation of transportin itself. Cells modified to express higher levels of CC3 have a slower rate of nuclear import and, as described earlier, show an increased sensitivity to death signals. A mutant CC3 protein lacking proapoptotic activity has a lower affinity for transportin, is displaced from it by RanGTP, and fails to inhibit nuclear import in vitro and in vivo. Together, our results support a correlation between the ability of CC3 to form a RanGTP-resistant complex with importins, inhibit nuclear import, and induce apoptosis. Significantly, a dominant-negative form of importin beta1 shown previously to inhibit multiple transport pathways induces rapid cell death, strongly indicating that inhibition of nuclear transport serves as a potent apoptotic signal.

AHNAK interacts with the DNA ligase IV-XRCC4 complex and stimulates DNA ligase IV-mediated double-stranded ligation.

AHNAK is a high molecular weight protein that is under-expressed in several radiosensitive neuroblastoma cell lines. Using immunoaffinity purification or purified proteins, we show that AHNAK interacts specifically with the DNA ligase IV-XRCC4 complex, a complex that functions in DNA non-homologous end-joining. Furthermore, AHNAK and the DNA ligase IV-XRCC4 complex co-immunoprecipitate demonstrating an in vivo interaction. This interaction is specific and is not observed with other DNA ligases nor with other components of the DNA non-homologous end-joining machinery. We characterised AHNAK as a protein that stimulates the double-stranded (DS) ligation activity of DNA ligase IV-XRCC4. We show that AHNAK has weak DNA-binding activity and forms a stable complex with the DNA ligase IV-XRCC4 complex on DNA. AHNAK is also able to link two DNA molecules to a similar extent to that previously reported for Ku. Together, these findings demonstrate new activities for AHNAK, and raise the possibility that it may function to modulate DNA non-homologous end-joining.

Inhibition of Chk1 by activated PKB/Akt.

We have shown recently that DNA damage effector kinase Chk1 is phosphorylated in vitro by protein kinase B/Akt (PKB/Akt) on serine 280. Activation of Chk1 by DNA damage in vivo is suppressed in presence of activated PKB. In this study we show that Chk1 is phosphorylated by PKB in vivo, and that increased phosphorylation by PKB on serine 280 correlates with impairment of Chk1 activation by DNA damage. Our results indicate a likely mechanism for the negative effects that phosphorylation of serine 280 has on activation of Chk1. The Chk1 protein phosphorylated by PKB on serine 280 does not enter into protein complexes after replication arrest. Moreover, Chk1 phosphorylated by PKB fails to undergo activating phosphorylation on serine 345 by ATM/ATR. Phosphorylation by ATM/ATR and association with other checkpoint proteins are essential steps in activation of Chk1. Inhibition of these steps provides a plausible explanation for the observed attenuation of Chk1 activation by activated PKB after DNA damage.

Promotion of mitosis by activated protein kinase B after DNA damage involves polo-like kinase 1 and checkpoint protein CHFR.

The role of the protein kinase B (PKB/Akt) in the regulation of cell survival and proliferation is well established. PKB is a key effector in the phosphatidylinositol 3-kinase pathway and plays a role in the initiation of S phase and in the G(2)-M transition. I report here that activated PKB shortens the G(2) arrest induced by DNA damage and promotes early entry into mitosis. Activated PKB supports high levels of expression and activity of the polo-like kinase 1 (Plk1) after DNA damage as cells accumulate in G(2). The checkpoint protein CHFR implicated in degradation of Plk1 is involved in the regulation of Plk1 by PKB. PKB phosphorylates CHFR in vitro and in vivo. Expression of a mutant form of CHFR that cannot be phosphorylated by PKB results in reduction of levels of Plk1 and inhibition of mitotic entry under normal conditions and after DNA damage. Results of this study support a model in which PKB facilitates mitotic resolution of DNA damage-induced G(2) arrest by inhibiting the checkpoint function of CHFR. The deregulated activation of PKB that occurs frequently in tumors might inhibit CHFR activity after DNA damage and therefore promote Plk1 accumulation leading to the disruption of the DNA damage checkpoint.

A role for PI 3-kinase and PKB activity in the G2/M phase of the cell cycle.

The role of the PI 3-kinase cascade in regulation of cell growth is well established [1]. PKB (protein kinase B) is a key downstream effector of the PI 3-kinase pathway and is best known for its antiapoptotic effects [2,3] and the role it plays in initiation of S phase [4]. Here, we show that PKB activity is high in the G2/M phase of the cell cycle in epithelial cells. Inhibition of the PI 3-kinase pathway in MDCK cells induces apoptosis at the G2/M transition, prevents activation of cyclin B-associated kinase, and prohibits entry of the surviving cells into mitosis. All of these consequences of the inhibition of PI 3-kinase are relieved by expression of a constitutively active form of PKB (caPKB), indicating that PKB plays a role in regulation of the G2/M phase. Inhibition of PI 3-kinase results in activation of Chk1, whereas caPKB inhibits the ability of Chk1 to become activated in response to treatment with hydroxyurea. Preliminary data show that PKB phosphorylates the Chk1 polypeptide in vitro on serine 280. These results not only implicate PKB activity in transition through the G2/M stage of the cell cycle, but they also suggest the existence of crosstalk between the PI 3-kinase pathway and the key regulators of the DNA damage checkpoint machinery.