There is a strong rationale to therapeutically target the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway in breast cancer since it is highly deregulated in this disease and it also mediates resistance to anti-HER2 therapies. However, initial studies with rapalogs, allosteric inhibitors of mTORC1, have resulted in limited clinical efficacy probably due to the release of a negative regulatory feedback loop that triggers AKT and ERK signaling. Since activation of AKT occurs via PI3K, we decided to explore whether PI3K inhibitors prevent the activation of these compensatory pathways. Using HER2-overexpressing breast cancer cells as a model, we observed that PI3K inhibitors abolished AKT activation. However, PI3K inhibition resulted in a compensatory activation of the ERK signaling pathway. This enhanced ERK signaling occurred as a result of activation of HER family receptors as evidenced by induction of HER receptors dimerization and phosphorylation, increased expression of HER3 and binding of adaptor molecules to HER2 and HER3. The activation of ERK was prevented with either MEK inhibitors or anti-HER2 monoclonal antibodies and tyrosine kinase inhibitors. Combined administration of PI3K inhibitors with either HER2 or MEK inhibitors resulted in decreased proliferation, enhanced cell death and superior anti-tumor activity compared with single agent PI3K inhibitors. Our findings indicate that PI3K inhibition in HER2-overexpressing breast cancer activates a new compensatory pathway that results in ERK dependency. Combined anti-MEK or anti-HER2 therapy with PI3K inhibitors may be required in order to achieve optimal efficacy in HER2-overexpressing breast cancer. This approach warrants clinical evaluation.

In advanced cancer, including glioblastoma, the transforming growth factor β (TGF-β) pathway acts as an oncogenic factor and is considered to be a therapeutic target. Using a functional RNAi screen, we identified the deubiquitinating enzyme ubiquitin-specific peptidase 15 (USP15) as a key component of the TGF-β signaling pathway. USP15 binds to the SMAD7-SMAD specific E3 ubiquitin protein ligase 2 (SMURF2) complex and deubiquitinates and stabilizes type I TGF-β receptor (TβR-I), leading to an enhanced TGF-β signal. High expression of USP15 correlates with high TGF-β activity, and the USP15 gene is found amplified in glioblastoma, breast and ovarian cancer. USP15 amplification confers poor prognosis in individuals with glioblastoma. Downregulation or inhibition of USP15 in a patient-derived orthotopic mouse model of glioblastoma decreases TGF-β activity. Moreover, depletion of USP15 decreases the oncogenic capacity of patient-derived glioma-initiating cells due to the repression of TGF-β signaling. Our results show that USP15 regulates the TGF-β pathway and is a key factor in glioblastoma pathogenesis.

Small molecule inhibitors of HER2 are clinically active in women with advanced HER2 positive breast cancer who have progressed on trastuzumab treatment. However, the effectiveness of this class of agents is limited by either primary resistance or acquired resistance. Using an unbiased genetic approach we performed a genome wide loss-of-function shRNA screen to identify novel modulators of resistance to lapatinib, a recently approved anti-HER2 tyrosine kinase inhibitor. Here, we have identified the tumour suppressor PTEN as a modulator of lapatinib sensitivity in vitro and in vivo. In addition, we demonstrate that two dominant activating mutations in PIK3CA (E545K and H1047R), which are prevalent in breast cancer, also confer resistance to lapatinib. Furthermore, we show that PI3K induced lapatinib resistance can be abrogated through the use of NVP-BEZ235, a dual inhibitor of PI3K/mTOR. Our data show that deregulation of the PI3K pathway, either through loss-of-function mutations in PTEN or dominant activating mutations in PIK3CA, leads to lapatinib resistance which can be effectively reversed by NVP-BEZ235.

Synthetic biology is a promising tool to study the function and properties of gene regulatory networks. Gene circuits with predefined behaviours have been successfully built and modelled, but largely on a case-by-case basis. Here we go beyond individual networks and explore both computationally and synthetically the design space of possible dynamical mechanisms for 3-node stripe-forming networks. First, we computationally test every possible 3-node network for stripe formation in a morphogen gradient. We discover four different dynamical mechanisms to form a stripe and identify the minimal network of each group. Next, with the help of newly established engineering criteria we build these four networks synthetically and show that they indeed operate with four fundamentally distinct mechanisms. Finally, this close match between theory and experiment allows us to infer and subsequently build a 2-node network that represents the archetype of the explored design space.

Clinical benefits from trastuzumab and other anti-HER2 therapies in patients with HER2 amplified breast cancer remain limited by primary or acquired resistance. To identify potential mechanisms of resistance, we established trastuzumab-resistant HER2 amplified breast cancer cells by chronic exposure to trastuzumab treatment. Genomewide copy-number variation analyses of the resistant cells compared with parental cells revealed a focal amplification of genomic DNA containing the cyclin E gene. In a cohort of 34 HER2 + patients treated with trastuzumab-based therapy, we found that cyclin E amplification/overexpression was associated with a worse clinical benefit (33.3% compared with 87.5%, P < 0.02) and a lower progression-free survival (6 mo vs. 14 mo, P < 0.002) compared with nonoverexpressing cyclin E tumors. To dissect the potential role of cyclin E in trastuzumab resistance, we studied the effects of cyclin E overexpression and cyclin E suppression. Cyclin E overexpression resulted in resistance to trastuzumab both in vitro and in vivo. Inhibition of cyclin E activity in cyclin E-amplified trastuzumab resistant clones, either by knockdown of cyclin E expression or treatment with cyclin-dependent kinase 2 (CDK2) inhibitors, led to a dramatic decrease in proliferation and enhanced apoptosis. In vivo, CDK2 inhibition significantly reduced tumor growth of trastuzumab-resistant xenografts. Our findings point to a causative role for cyclin E overexpression and the consequent increase in CDK2 activity in trastuzumab resistance and suggest that treatment with CDK2 inhibitors may be a valid strategy in patients with breast tumors with HER2 and cyclin E coamplification/overexpression. HER2 is a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, which includes EGFR itself, HER2, HER3, and HER4. Homo-or heterodimerization of these receptors results in phosphorylation of residues in the intracellular domain and consequent recruitment of adapter molecules responsible for the initiation of several signaling pathways involved in cell proliferation and survival (1, 2). Approximately 20% of breast cancers exhibit HER2 gene amplification/overexpression, resulting in an aggressive tumor phenotype and reduced survival (3, 4). Therapy of HER2 + breast cancer with anti-HER2 agents, including monoclonal antibodies and small molecule tyrosine kinase inhibitors, has markedly improved the outcome of this disease (5). Trastuzumab, a recombinant humanized monoclonal antibody that binds to the extracellular domain of HER2, improves survival in patients with HER2 + breast cancer, in both the metastatic (6, 7) and adjuvant settings (8). The overall antitumor activity of trastuzumab is due to a combination of mechanisms, including inhibition of ligandindependent HER2 dimerization (9), HER2 down-regulation (10,11), that lead to disruption of HER2-dependent PI3K/Akt signaling (12) and induction of G1 arrest through stabilization of the CDK inhibitor p27 (13). In addition, trastuzumab also mediates antibod...

Synthetic biology has developed numerous parts for building synthetic gene circuits. However, few parts have been described for prokaryotes to integrate two signals at a promoter in an AND fashion, i.e. the promoter is only activated in the presence of both signals. Here we present a new part for this function: a split intein T7 RNA polymerase. We divide T7 RNA polymerase into two expression domains and fuse each to a split intein. Only when both domains are expressed does the split intein mediate protein trans-splicing, yielding a full-length T7 RNA polymerase that can transcribe genes via a T7 promoter. We demonstrate an AND gate with the new part: the signal-to-background ratio is very high, resulting in an almost digital signal. This has utility for more complex circuits and so we construct a band-pass filter in Escherichia coli. The split intein approach should be widely applicable for engineering artificial gene circuit parts.

A significant number of neurofibromatosis type 1 (NF1) mutations result in exon skipping. The majority of these mutations do not occur in the canonical splice sites and can produce different aberrant transcripts whose proportions have not been well studied. It has been hypothesized that differences in the mutation-determined NF1-transcriptional profile could partially explain disease variability among patients bearing the same NF1 splice defect. In order to gain insight into these aspects, we analyzed the proportion of the different transcripts generated by nine NF1-splicing mutations in 30 patients. We assessed the influence of the mutation in the NF1-related transcriptional profiles and investigated the existence of individual differences in a global manner. We analyzed potential differences in tissue-specific transcriptional profiles and evaluated the influence of sample processing and mRNA nonsense-mediated decay (NMD). Small transcriptional differences were found in neurofibromas and neurofibroma-derived Schwann cells (SC) compared to blood. We also detected a higher cell culture-dependent NMD. We observed that mutation per se explains 93.5% of the profile variability among mutations studied. However, despite the importance of mutation in determining the proportion of NF1 transcripts generated, we found certain variability among patients with the same mutation. From our results, it seems that genetic factors influencing RNA processing play a minor role in determining the NF1-transcriptional profile. Nevertheless neurofibromin studies would clarify whether these small differences translate into significant functional changes that could explain the great clinical expressivity observed in the disease or any of the disease-related traits.

The neurofibromatosis type 1 gene has one of the highest mutation rates in humans: about 50% of NF1 patients are de novo cases. Although direct mutation characterization has greatly improved over the past decade, in the context of clinical genetics services worldwide, there is still a significant number of patients for which, while fulfilling NF1 clinical criteria, no constitutive mutation is found at a desired time. This is particularly critical for prenatal genetic testing of sporadic cases. Here we describe the use of loss of heterozygosity information in neurofibromas to obtain linkage information on the affected NF1 haplotype, which may be applied for prenatal testing in sporadic patients. However, proper genetic counseling should be provided regarding the possibility of somatic mosaicism.