OBJECTIVE

All patients with EGF receptor (EGFR)-mutant lung cancers eventually develop acquired resistance to EGFR tyrosine kinase inhibitors (TKI). Smaller series have identified various mechanisms of resistance, but systematic evaluation of a large number of patients to definitively establish the frequency of various mechanisms has not been conducted.

METHODS

Patients with lung adenocarcinomas and acquired resistance to erlotinib or gefitinib enrolled onto a prospective biopsy protocol and underwent a rebiopsy after the development of acquired resistance. Histology was reviewed. Samples underwent genotyping for mutations in EGFR, AKT1, BRAF, ERBB2, KRAS, MEK1, NRAS and PIK3CA, and FISH for MET and HER2.

RESULTS

Adequate tumor samples for molecular analysis were obtained in 155 patients. Ninety-eight had second-site EGFR T790M mutations [63%; 95% confidence interval (CI), 55%-70%] and four had small cell transformation (3%, 95% CI, 0%-6%). MET amplification was seen in 4 of 75 (5%; 95% CI, 1%-13%). HER2 amplification was seen in 3 of 24 (13%; 95% CI, 3%-32%). We did not detect any acquired mutations in PIK3CA, AKT1, BRAF, ERBB2, KRAS, MEK1, or NRAS (0 of 88, 0%; 95% CI, 0%-4%). Overlap among mechanisms of acquired resistance was seen in 4%.

CONCLUSIONS

This is the largest series reporting mechanisms of acquired resistance to EGFR-TKI therapy. We identified EGFR T790M as the most common mechanism of acquired resistance, whereas MET amplification, HER2 amplification, and small cell histologic transformation occur less frequently. More comprehensive methods to characterize molecular alterations in this setting are needed to improve our understanding of acquired resistance to EGFR-TKIs.

Although AKT1 (v-akt murine thymoma viral oncogene homologue 1) kinase is a central member of possibly the most frequently activated proliferation and survival pathway in cancer, mutation of AKT1 has not been widely reported. Here we report the identification of a somatic mutation in human breast, colorectal and ovarian cancers that results in a glutamic acid to lysine substitution at amino acid 17 (E17K) in the lipid-binding pocket of AKT1. Lys 17 alters the electrostatic interactions of the pocket and forms new hydrogen bonds with a phosphoinositide ligand. This mutation activates AKT1 by means of pathological localization to the plasma membrane, stimulates downstream signalling, transforms cells and induces leukaemia in mice. This mechanism indicates a direct role of AKT1 in human cancer, and adds to the known genetic alterations that promote oncogenesis through the phosphatidylinositol-3-OH kinase/AKT pathway. Furthermore, the E17K substitution decreases the sensitivity to an allosteric kinase inhibitor, so this mutation may have important clinical utility for AKT drug development.

Akt/PKB is a serine/threonine protein kinase that functions as a critical regulator of cell survival and proliferation. Akt/PKB family comprises three highly homologous members known as PKBalpha/Akt1, PKBbeta/Akt2 and PKBgamma/Akt3 in mammalian cells. Similar to many other protein kinases, Akt/PKB contains a conserved domain structure including a specific PH domain, a central kinase domain and a carboxyl-terminal regulatory domain that mediates the interaction between signaling molecules. Akt/PKB plays important roles in the signaling pathways in response to growth factors and other extracellular stimuli to regulate several cellular functions including nutrient metabolism, cell growth, apoptosis and survival. This review surveys recent developments in understanding the molecular mechanisms of Akt/PKB activation and its roles in cell survival in normal and cancer cells.

Breast carcinoma is the leading cause of cancer-related mortality in women worldwide, with an estimated 1.38 million new cases and 458,000 deaths in 2008 alone. This malignancy represents a heterogeneous group of tumours with characteristic molecular features, prognosis and responses to available therapy. Recurrent somatic alterations in breast cancer have been described, including mutations and copy number alterations, notably ERBB2 amplifications, the first successful therapy target defined by a genomic aberration. Previous DNA sequencing studies of breast cancer genomes have revealed additional candidate mutations and gene rearrangements. Here we report the whole-exome sequences of DNA from 103 human breast cancers of diverse subtypes from patients in Mexico and Vietnam compared to matched-normal DNA, together with whole-genome sequences of 22 breast cancer/normal pairs. Beyond confirming recurrent somatic mutations in PIK3CA, TP53, AKT1, GATA3 and MAP3K1, we discovered recurrent mutations in the CBFB transcription factor gene and deletions of its partner RUNX1. Furthermore, we have identified a recurrent MAGI3-AKT3 fusion enriched in triple-negative breast cancer lacking oestrogen and progesterone receptors and ERBB2 expression. The MAGI3-AKT3 fusion leads to constitutive activation of AKT kinase, which is abolished by treatment with an ATP-competitive AKT small-molecule inhibitor.

Transplantation of adult bone marrow-derived mesenchymal stem cells has been proposed as a strategy for cardiac repair following myocardial damage. However, poor cell viability associated with transplantation has limited the reparative capacity of these cells in vivo. In this study, we genetically engineered rat mesenchymal stem cells using ex vivo retroviral transduction to overexpress the prosurvival gene Akt1 (encoding the Akt protein). Transplantation of 5 x 10(6) cells overexpressing Akt into the ischemic rat myocardium inhibited the process of cardiac remodeling by reducing intramyocardial inflammation, collagen deposition and cardiac myocyte hypertrophy, regenerated 80-90% of lost myocardial volume, and completely normalized systolic and diastolic cardiac function. These observed effects were dose (cell number) dependent. Mesenchymal stem cells transduced with Akt1 restored fourfold greater myocardial volume than equal numbers of cells transduced with the reporter gene lacZ. Thus, mesenchymal stem cells genetically enhanced with Akt1 can repair infarcted myocardium, prevent remodeling and nearly normalize cardiac performance.

Maintenance of the blood system is dependent on dormant haematopoietic stem cells (HSCs) with long-term self-renewal capacity. After injury these cells are induced to proliferate to quickly re-establish homeostasis. The signalling molecules promoting the exit of HSCs out of the dormant stage remain largely unknown. Here we show that in response to treatment of mice with interferon-alpha (IFNalpha), HSCs efficiently exit G(0) and enter an active cell cycle. HSCs respond to IFNalpha treatment by the increased phosphorylation of STAT1 and PKB/Akt (also known as AKT1), the expression of IFNalpha target genes, and the upregulation of stem cell antigen-1 (Sca-1, also known as LY6A). HSCs lacking the IFNalpha/beta receptor (IFNAR), STAT1 (ref. 3) or Sca-1 (ref. 4) are insensitive to IFNalpha stimulation, demonstrating that STAT1 and Sca-1 mediate IFNalpha-induced HSC proliferation. Although dormant HSCs are resistant to the anti-proliferative chemotherapeutic agent 5-fluoro-uracil, HSCs pre-treated (primed) with IFNalpha and thus induced to proliferate are efficiently eliminated by 5-fluoro-uracil exposure in vivo. Conversely, HSCs chronically activated by IFNalpha are functionally compromised and are rapidly out-competed by non-activatable Ifnar(-/-) cells in competitive repopulation assays. Whereas chronic activation of the IFNalpha pathway in HSCs impairs their function, acute IFNalpha treatment promotes the proliferation of dormant HSCs in vivo. These data may help to clarify the so far unexplained clinical effects of IFNalpha on leukaemic cells, and raise the possibility for new applications of type I interferons to target cancer stem cells.

Protein kinase B or Akt (PKB/Akt) is a serine/threonine kinase, which in mammals comprises three highly homologous members known as PKBalpha (Akt1), PKBbeta (Akt2), and PKBgamma (Akt3). PKB/Akt is activated in cells exposed to diverse stimuli such as hormones, growth factors, and extracellular matrix components. The activation mechanism remains to be fully characterised but occurs downstream of phosphoinositide 3-kinase (PI-3K). PI-3K generates phosphatidylinositol-3,4,5-trisphosphate (PIP(3)), a lipid second messenger essential for the translocation of PKB/Akt to the plasma membrane where it is phosphorylated and activated by phosphoinositide-dependent kinase-1 (PDK-1) and possibly other kinases. PKB/Akt phosphorylates and regulates the function of many cellular proteins involved in processes that include metabolism, apoptosis, and proliferation. Recent evidence indicates that PKB/Akt is frequently constitutively active in many types of human cancer. Constitutive PKB/Akt activation can occur due to amplification of PKB/Akt genes or as a result of mutations in components of the signalling pathway that activates PKB/Akt. Although the mechanisms have not yet been fully characterised, constitutive PKB/Akt signalling is believed to promote proliferation and increased cell survival and thereby contributing to cancer progression. This review surveys recent developments in understanding the mechanisms and consequences of PKB/Akt activation in human malignancy.

Phosphatidylinositol 3-kinase (PI3K)/AKT pathway aberrations are common in cancer. By applying mass spectroscopy-based sequencing and reverse-phase protein arrays to 547 human breast cancers and 41 cell lines, we determined the subtype specificity and signaling effects of PIK3CA, AKT, and PTEN mutations and the effects of PIK3CA mutations on responsiveness to PI3K inhibition in vitro and on outcome after adjuvant tamoxifen. PIK3CA mutations were more common in hormone receptor-positive (34.5%) and HER2-positive (22.7%) than in basal-like tumors (8.3%). AKT1 (1.4%) and PTEN (2.3%) mutations were restricted to hormone receptor-positive cancers. Unlike AKT1 mutations that were absent from cell lines, PIK3CA (39%) and PTEN (20%) mutations were more common in cell lines than tumors, suggesting a selection for these but not AKT1 mutations during adaptation to culture. PIK3CA mutations did not have a significant effect on outcome after adjuvant tamoxifen therapy in 157 hormone receptor-positive breast cancer patients. PIK3CA mutations, in comparison with PTEN loss and AKT1 mutations, were associated with significantly less and inconsistent activation of AKT and of downstream PI3K/AKT signaling in tumors and cell lines. PTEN loss and PIK3CA mutation were frequently concordant, suggesting different contributions to pathophysiology. PTEN loss rendered cells significantly more sensitive to growth inhibition by the PI3K inhibitor LY294002 than did PIK3CA mutations. Thus, PI3K pathway aberrations likely play a distinct role in the pathogenesis of different breast cancer subtypes. The specific aberration present may have implications for the selection of PI3K-targeted therapies in hormone receptor-positive breast cancer.

Human immunodeficiency virus (HIV)-1 depends on the host cell machinery to support its replication. To discover cellular factors associated with HIV-1 replication, we conducted a genome-scale siRNA screen, revealing more than 311 host factors, including 267 that were not previously linked to HIV. Surprisingly, there was little overlap between these genes and the HIV dependency factors described recently. However, an analysis of the genes identified in both screens revealed overlaps in several of the associated pathways or protein complexes, including the SP1/mediator complex and the NF-kappaB signaling pathway. cDNAs for a subset of the identified genes were used to rescue HIV replication following knockdown of the cellular mRNA providing strong evidence that the following six genes are previously uncharacterized host factors for HIV: AKT1, PRKAA1, CD97, NEIL3, BMP2K, and SERPINB6. This study highlights both the power and shortcomings of large scale loss-of-function screens in discovering host-pathogen interactions.

The serine-threonine kinase Akt, also known as protein kinase B (PKB), is an important effector for phosphatidylinositol 3-kinase signaling initiated by numerous growth factors and hormones. Akt2/PKBbeta, one of three known mammalian isoforms of Akt/PKB, has been demonstrated recently to be required for at least some of the metabolic actions of insulin (Cho, H., Mu, J., Kim, J. K., Thorvaldsen, J. L., Chu, Q., Crenshaw, E. B., Kaestner, K. H., Bartolomei, M. S., Shulman, G. I., and Birnbaum, M. J. (2001) Science 292, 1728-1731). Here we show that mice deficient in another closely related isoform of the kinase, Akt1/PKBalpha, display a conspicuous impairment in organismal growth. Akt1(-/-) mice demonstrated defects in both fetal and postnatal growth, and these persisted into adulthood. However, in striking contrast to Akt2/PKBbeta null mice, Akt1/PKBalpha-deficient mice are normal with regard to glucose tolerance and insulin-stimulated disposal of blood glucose. Thus, the characterization of the Akt1 knockout mice and its comparison to the previously reported Akt2 deficiency phenotype reveals the non-redundant functions of Akt1 and Akt2 genes with respect to organismal growth and insulin-regulated glucose metabolism.

Treatment decisions for patients with lung cancer have historically been based on tumour histology. Some understanding of the molecular composition of tumours has led to the development of targeted agents, for which initial findings are promising. Clearer understanding of mutations in relevant genes and their effects on cancer cell proliferation and survival, is, therefore, of substantial interest. We review current knowledge about molecular subsets in non-small-cell lung cancer that have been identified as potentially having clinical relevance to targeted therapies. Since mutations in EGFR and KRAS have been extensively reviewed elsewhere, here, we discuss subsets defined by so-called driver mutations in ALK, HER2 (also known as ERBB2), BRAF, PIK3CA, AKT1, MAP2K1, and MET. The adoption of treatment tailored according to the genetic make-up of individual tumours would involve a paradigm shift, but might lead to substantial therapeutic improvements.

Loss of PTEN function leads to activation of phosphoinositide 3-kinase (PI3K) signaling and Akt. Clinical trials are now testing whether mammalian target of rapamycin (mTOR) inhibition is useful in treating PTEN-null cancers. Here, we report that mTOR inhibition induced apoptosis of epithelial cells and the complete reversal of a neoplastic phenotype in the prostate of mice expressing human AKT1 in the ventral prostate. Induction of cell death required the mitochondrial pathway, as prostate-specific coexpression of BCL2 blocked apoptosis. Thus, there is an mTOR-dependent survival signal required downstream of Akt. Bcl2 expression, however, only partially restored intraluminal cell growth in the setting of mTOR inhibition. Expression profiling showed that Hif-1 alpha targets, including genes encoding most glycolytic enzymes, constituted the dominant transcriptional response to AKT activation and mTOR inhibition. These data suggest that the expansion of AKT-driven prostate epithelial cells requires mTOR-dependent survival signaling and activation of HIF-1 alpha, and that clinical resistance to mTOR inhibitors may emerge through BCL2 expression and/or upregulation of HIF-1 alpha activity.

OBJECTIVE

Targeting oncogenic drivers (genomic alterations critical to cancer development and maintenance) has transformed the care of patients with lung adenocarcinomas. The Lung Cancer Mutation Consortium was formed to perform multiplexed assays testing adenocarcinomas of the lung for drivers in 10 genes to enable clinicians to select targeted treatments and enroll patients into clinical trials.

OBJECTIVE

To determine the frequency of oncogenic drivers in patients with lung adenocarcinomas and to use the data to select treatments targeting the identified driver(s) and measure survival.

METHODS

From 2009 through 2012, 14 sites in the United States enrolled patients with metastatic lung adenocarcinomas and a performance status of 0 through 2 and tested their tumors for 10 drivers. Information was collected on patients, therapies, and survival.

METHODS

Tumors were tested for 10 oncogenic drivers, and results were used to select matched targeted therapies.

METHODS

Determination of the frequency of oncogenic drivers, the proportion of patients treated with genotype-directed therapy, and survival.

RESULTS

From 2009 through 2012, tumors from 1007 patients were tested for at least 1 gene and 733 for 10 genes (patients with full genotyping). An oncogenic driver was found in 466 of 733 patients (64%). Among these 733 tumors, 182 tumors (25%) had the KRAS driver; sensitizing EGFR, 122 (17%); ALK rearrangements, 57 (8%); other EGFR, 29 (4%); 2 or more genes, 24 (3%); ERBB2 (formerly HER2), 19 (3%); BRAF, 16 (2%); PIK3CA, 6 (<1%); MET amplification, 5 (<1%); NRAS, 5 (<1%); MEK1, 1 (<1%); AKT1, 0. Results were used to select a targeted therapy or trial in 275 of 1007 patients (28%). The median survival was 3.5 years (interquartile range [IQR], 1.96-7.70) for the 260 patients with an oncogenic driver and genotype-directed therapy compared with 2.4 years (IQR, 0.88-6.20) for the 318 patients with any oncogenic driver(s) who did not receive genotype-directed therapy (propensity score-adjusted hazard ratio, 0.69 [95% CI, 0.53-0.9], P = .006).

CONCLUSIONS

Actionable drivers were detected in 64% of lung adenocarcinomas. Multiplexed testing aided physicians in selecting therapies. Although individuals with drivers receiving a matched targeted agent lived longer, randomized trials are required to determine if targeting therapy based on oncogenic drivers improves survival.

BACKGROUND

clinicaltrials.gov Identifier: NCT01014286.

The androgen receptor (AR) plays a key role in progression to incurable androgen ablation-resistant prostate cancer (PCA). We have identified three novel AR splice variants lacking the ligand-binding domain (designated as AR3, AR4, and AR5) in hormone-insensitive PCA cells. AR3, one of the major splice variants expressed in human prostate tissues, is constitutively active, and its transcriptional activity is not regulated by androgens or antiandrogens. Immunohistochemistry analysis on tissue microarrays containing 429 human prostate tissue samples shows that AR3 is significantly up-regulated during PCA progression and AR3 expression level is correlated with the risk of tumor recurrence after radical prostatectomy. Overexpression of AR3 confers ablation-independent growth of PCA cells, whereas specific knockdown of AR3 expression (without altering AR level) in hormone-resistant PCA cells attenuates their growth under androgen-depleted conditions in both cell culture and xenograft models, suggesting an indispensable role of AR3 in ablation-independent growth of PCA cells. Furthermore, AR3 may play a distinct, yet essential, role in ablation-independent growth through the regulation of a unique set of genes, including AKT1, which are not regulated by the prototype AR. Our data suggest that aberrant expression of AR splice variants may be a novel mechanism underlying ablation independence during PCA progression, and AR3 may serve as a prognostic marker to predict patient outcome in response to hormonal therapy. Given that these novel AR splice variants are not inhibited by currently available antiandrogen drugs, development of new drugs targeting these AR isoforms may potentially be effective for treatment of ablation-resistant PCA.

Akt/protein kinase B critically regulates the balance between cell survival and apoptosis. Phosphorylation of Akt at two key sites, the activation loop and the hydrophobic motif, activates the kinase and promotes cell survival. The mechanism of dephosphorylation and signal termination is unknown. Here, we identify a protein phosphatase, PH domain leucine-rich repeat protein phosphatase (PHLPP), that specifically dephosphorylates the hydrophobic motif of Akt (Ser473 in Akt1), triggering apoptosis and suppressing tumor growth. The effects of PHLPP on apoptosis are prevented in cells expressing an S473D construct of Akt, revealing that the hydrophobic motif is the primary cellular target of PHLPP. PHLPP levels are markedly reduced in several colon cancer and glioblastoma cell lines that have elevated Akt phosphorylation. Reintroduction of PHLPP into a glioblastoma cell line causes a dramatic suppression of tumor growth. These data are consistent with PHLPP terminating Akt signaling by directly dephosphorylating and inactivating Akt.

Although increased external load initially induces cardiac hypertrophy with preserved contractility, sustained overload eventually leads to heart failure through poorly understood mechanisms. Here we describe a conditional transgenic system in mice characterized by the sequential development of adaptive cardiac hypertrophy with preserved contractility in the acute phase and dilated cardiomyopathy in the chronic phase following the induction of an activated Akt1 gene in the heart. Coronary angiogenesis was enhanced during the acute phase of adaptive cardiac growth but reduced as hearts underwent pathological remodeling. Enhanced angiogenesis in the acute phase was associated with mammalian target of rapamycin-dependent induction of myocardial VEGF and angiopoietin-2 expression. Inhibition of angiogenesis by a decoy VEGF receptor in the acute phase led to decreased capillary density, contractile dysfunction, and impaired cardiac growth. Thus, both heart size and cardiac function are angiogenesis dependent, and disruption of coordinated tissue growth and angiogenesis in the heart contributes to the progression from adaptive cardiac hypertrophy to heart failure.

The serine/threonine kinase Akt has been implicated in the control of cell survival and metabolism. Here we report the disruption of the most ubiquitously expressed member of the akt family of genes, akt1, in the mouse. Akt1(-/-) mice are viable but smaller when compared to wild-type littermates. In addition, the life span of Akt1(-/-) mice, upon exposure to genotoxic stress, is shorter. However, Akt1(-/-) mice do not display a diabetic phenotype. Increased spontaneous apoptosis in testes, and attenuation of spermatogenesis is observed in Akt1(-/-) male mice. Increased spontaneous apoptosis is also observed in the thymi of Akt1(-/-) mice, and Akt1(-/-) thymocytes are more sensitive to apoptosis induced by gamma-irradiation and dexamethasone. Finally, Akt1(-/-) mouse embryo fibroblasts (MEFs) are more susceptible to apoptosis induced by TNF, anti-Fas, UV irradiation, and serum withdrawal.

AKT-GSK3beta signaling is a target of lithium and as such has been implicated in the pathogenesis of mood disorders. Here, we provide evidence that this signaling pathway also has a role in schizophrenia. Specifically, we present convergent evidence for a decrease in AKT1 protein levels and levels of phosphorylation of GSK3beta at Ser9 in the peripheral lymphocytes and brains of individuals with schizophrenia; a significant association between schizophrenia and an AKT1 haplotype associated with lower AKT1 protein levels; and a greater sensitivity to the sensorimotor gating-disruptive effect of amphetamine, conferred by AKT1 deficiency. Our findings support the proposal that alterations in AKT1-GSK3beta signaling contribute to schizophrenia pathogenesis and identify AKT1 as a potential schizophrenia susceptibility gene. Consistent with this proposal, we also show that haloperidol induces a stepwise increase in regulatory phosphorylation of AKT1 in the brains of treated mice that could compensate for an impaired function of this signaling pathway in schizophrenia.

To elucidate the functions of the serine/threonine kinase Akt/PKB in vivo, we generated mice lacking both akt1 and akt2 genes. Akt1/Akt2 double-knockout (DKO) mice exhibit severe growth deficiency and die shortly after birth. These mice display impaired skin development because of a proliferation defect, severe skeletal muscle atrophy because of a marked decrease in individual muscle cell size, and impaired bone development. These defects are strikingly similar to the phenotypes of IGF-1 receptor-deficient mice and suggest that Akt may serve as the most critical downstream effector of the IGF-1 receptor during development. In addition, Akt1/Akt2 DKO mice display impeded adipogenesis. Specifically, Akt1 and Akt2 are required for the induced expression of PPARgamma, the master regulator of adipogenesis, establishing a new essential role for Akt in adipocyte differentiation. Overall, the combined deletion of Akt1 and Akt2 establishes in vivo roles for Akt in cell proliferation, growth, and differentiation. These functions of Akt were uncovered despite the observed lower level of Akt activity mediated by Akt3 in Akt1/Akt2 DKO cells, suggesting that a critical threshold level of Akt activity is required to maintain normal cell proliferation, growth, and differentiation.

Akt (also known as protein kinase B or PKB) comprises three closely related isoforms Akt1, Akt2 and Akt3 (or PKBα/β/γ respectively). We have a very good understanding of the mechanisms by which Akt isoforms are activated by growth factors and other extracellular stimuli as well as by oncogenic mutations in key upstream regulatory proteins including Ras, PI3-kinase subunits and PTEN. There are also an ever increasing number of Akt substrates being identified that play a role in the regulation of the diverse array of biological effects of activated Akt; this includes the regulation of cell proliferation, survival and metabolism. Dysregulation of Akt leads to diseases of major unmet medical need such as cancer, diabetes, cardiovascular and neurological diseases. As a result there has been substantial investment in the development of small molecular Akt inhibitors that act competitively with ATP or phospholipid binding, or allosterically. In this review we will briefly discuss our current understanding of how Akt isoforms are regulated, the substrate proteins they phosphorylate and how this integrates with the role of Akt in disease. We will furthermore discuss the types of Akt inhibitors that have been developed and are in clinical trials for human cancer, as well as speculate on potential on-target toxicities, such as disturbances of heart and vascular function, metabolism, memory and mood, which should be monitored very carefully during clinical trial.

The recent discovery of cancer stem cells (CSCs) has played a pivotal role in changing our view of carcinogenesis and chemotherapy. Based on this concept, CSCs are responsible for the formation and growth of neoplastic tissue and are naturally resistant to chemotherapy, explaining why traditional chemotherapies can initially shrink a tumor but fails to eradicate it in full, allowing eventual recurrence. Recently, we identified a CSC population in hepatocellular carcinoma (HCC) characterized by their CD133 phenotype. However, the molecular mechanism by which it escapes conventional therapies remains unknown. Here, we examined the sensitivity of these cells to chemotherapeutic agents (doxorubicin and fluorouracil) and the possible mechanistic pathway by which resistance may be regulated. Purified CD133+ HCC cells isolated from human HCC cell line and xenograft mouse models survived chemotherapy in increased proportions relative to most tumor cells which lack the CD133 phenotype; the underlying mechanism of which required the preferential expression of survival proteins involved in the Akt/PKB and Bcl-2 pathway. Treatment of CD133+ HCC cells with an AKT1 inhibitor, specific to the Akt/PKB pathway, significantly reduced the expression of the survival proteins that was normally expressed endogenously. In addition, treatment of unsorted HCC cells with both anticancer drugs in vitro significantly enriched the CD133+ subpopulation. In conclusion, our results show that CD133+ HCC cells contribute to chemoresistance through preferential activation of Akt/PKB and Bcl-2 cell survival response. Targeting of this specific survival signaling pathway in CD133+ HCC CSCs may provide a novel therapeutic model for the disease.

The serine/threonine kinase Akt/PKB plays key roles in the regulation of cell growth, survival, and metabolism. It remains unclear, however, whether the functions of individual Akt/PKB isoforms are distinct. To investigate the function of Akt2/PKBbeta, mice lacking this isoform were generated. Both male and female Akt2/PKBbeta-null mice exhibit mild growth deficiency and an age-dependent loss of adipose tissue or lipoatrophy, with all observed adipose depots dramatically reduced by 22 weeks of age. Akt2/PKBbeta-deficient mice are insulin resistant with elevated plasma triglycerides. In addition, Akt2/PKBbeta-deficient mice exhibit fed and fasting hyperglycemia, hyperinsulinemia, glucose intolerance, and impaired muscle glucose uptake. In males, insulin resistance progresses to a severe form of diabetes accompanied by pancreatic beta cell failure. In contrast, female Akt2/PKBbeta-deficient mice remain mildly hyperglycemic and hyperinsulinemic until at least one year of age. Thus, Akt2/PKBbeta-deficient mice exhibit growth deficiency similar to that reported previously for mice lacking Akt1/PKBalpha, indicating that both Akt2/PKBbeta and Akt1/PKBalpha participate in the regulation of growth. The marked hyperglycemia and loss of pancreatic beta cells and adipose tissue in Akt2/PKBbeta-deficient mice suggest that Akt2/PKBbeta plays critical roles in glucose metabolism and the development or maintenance of proper adipose tissue and islet mass for which other Akt/PKB isoforms are unable to fully compensate.

The monoclonal antibodies (moAb) panitumumab and cetuximab target the epidermal growth factor receptor (EGFR) and have proven valuable for the treatment of metastatic colorectal cancer (mCRC). EGFR-mediated signaling involves two main intracellular cascades: on one side KRAS activates BRAF, which in turn triggers the mitogen-activated protein kinases. On the other, membrane localization of the lipid kinase PIK3CA counteracts PTEN and promotes AKT1 phosphorylation, thereby activating a parallel intracellular axis. Constitutive activation of KRAS bypasses the corresponding signaling cascade and, accordingly, patients with mCRC bearing KRAS mutations are clinically resistant to therapy with panitumumab or cetuximab. We hypothesized that mutations activating PIK3CA could also preclude responsiveness to EGFR-targeted moAbs through a similar mechanism. Here, we present the mutational analysis of PIK3CA and KRAS and evaluation of the PTEN protein status in a cohort of 110 patients with mCRC treated with anti-EGFR moAbs. We observed 15 (13.6%) PIK3CA and 32 (29.0%) KRAS mutations. PIK3CA mutations were significantly associated with clinical resistance to panitumumab or cetuximab; none of the mutated patients achieved objective response (P = 0.038). When only KRAS wild-type tumors were analyzed, the statistical correlation was stronger (P = 0.016). Patients with PIK3CA mutations displayed a worse clinical outcome also in terms of progression-free survival (P = 0.035). Our data indicate that PIK3CA mutations can independently hamper the therapeutic response to panitumumab or cetuximab in mCRC. When the molecular status of the PIK3CA/PTEN and KRAS pathways are concomitantly ascertained, up to 70% of mCRC patients unlikely to respond to EGFR moAbs can be identified.

Ovarian cancer is the leading cause of death from gynecological malignancy and the fourth leading cause of cancer death among American women, yet little is known about its molecular aetiology. Studies using comparative genomic hybridization (CGH) have revealed several regions of recurrent, abnormal, DNA sequence copy number that may encode genes involved in the genesis or progression of the disease. One region at 3q26 found to be increased in copy number in approximately 40% of ovarian and others cancers contains PIK3CA, which encodes the p110alpha catalytic subunit of phosphatidylinositol 3-kinase (PI3-kinase). The association between PIK3CA copy number and PI3-kinase activity makes PIK3CA a candidate oncogene because a broad range of cancer-related functions have been associated with PI3-kinase mediated signalling. These include proliferation, glucose transport and catabolism, cell adhesion, apoptosis, RAS signalling and oncogenic transformation. In addition, downstream effectors of PI3-kinase, AKT1 and AKT2, have been found to be amplified or activated in human tumours, including ovarian cancer. We show here that PIK3CA is frequently increased in copy number in ovarian cancers, that the increased copy number is associated with increased PIK3CA transcription, p110alpha protein expression and PI3-kinase activity and that treatment with the PI3-kinase inhibitor LY294002 decreases proliferation and increases apoptosis. Our observations suggest PIK3CA is an oncogene that has an important role in ovarian cancer.