Physiological expression of PI3K H1047R mutation reveals its anti-metastatic potential in ErbB2-driven breast cancer.

p110α is a catalytic subunit of phosphoinositide 3-kinase (PI3K), a major downstream effector of receptor tyrosine kinase ErbB2, that is amplified and overexpressed in 20-30% of breast cancers, 40% of which have an activating mutation in p110α. Despite the high frequency of PIK3CA gain-of-function mutations, their prognostic value is controversial. Here, we employ a knock-in transgenic strategy to restrict the expression of an activated form of ErbB2 and p110α kinase domain mutation (p110αHR) in the mammary epithelium. Physiological levels of transgene expression under the control of their endogenous promoters did not result in a major synergistic effect. However, tumors arising in ErbB2/p110αHR bi-genic strain metastasized to the lung with significantly reduced capacity compared to tumors expressing ErbB2 alone. The reduced metastasis was further associated with retention of the myoepithelial layer reminiscent of ductal carcinoma in situ (DCIS), a non-invasive stage of human breast cancer. Molecular and biochemical analyses revealed that these poorly metastatic tumors exhibited a significant decrease in phospho-myosin light chain 2 (MLC2) associated with cellular contractility and migration. Examination of human samples for MLC2 activity revealed a progressive increase in cellular contractility between non-invasive DCIS and invasive ductal carcinoma. Collectively, these data argue that p110αHR mutation attenuates metastatic behavior in the context of ErbB2-driven breast cancer.

Elevated V-ATPase Activity Following PTEN Loss Is Required for Enhanced Oncogenic Signaling in Breast Cancer.

PTEN loss-of-function contributes to hyperactivation of the PI3K pathway and to drug resistance in breast cancer. Unchecked PI3K pathway signaling increases activation of the mechanistic target of rapamycin complex 1 (mTORC1), which promotes tumorigenicity. Several studies have suggested that vacuolar (H+)-ATPase (V-ATPase) complex activity is regulated by PI3K signaling. In this study, we showed that loss of PTEN elevated V-ATPase activity. Enhanced V-ATPase activity was mediated by increased expression of the ATPase H+ transporting accessory protein 2 (ATP6AP2), also known as the prorenin receptor (PRR). PRR is cleaved into a secreted extracellular fragment (sPRR) and an intracellular fragment (M8.9) that remains associated with the V-ATPase complex. Reduced PTEN expression increased V-ATPase complex activity in a PRR-dependent manner. Breast cancer cell lines with reduced PTEN expression demonstrated increased PRR expression. Similarly, PRR expression became elevated upon PTEN deletion in a mouse model of breast cancer. Interestingly, concentration of sPRR was elevated in the plasma of patients with breast cancer and correlated with tumor burden in HER2-enriched cancers. Moreover, PRR was essential for proper HER2 receptor expression, localization, and signaling. PRR knockdown attenuated HER2 signaling and resulted in reduced Akt and ERK 1/2 phosphorylation, and in lower mTORC1 activity. Overall, our study demonstrates a mechanism by which PTEN loss in breast cancer can potentiate multiple signaling pathways through upregulation of the V-ATPase complex. IMPLICATIONS: Our study contributed to the understanding of the role of the V-ATPase complex in breast cancer cell tumorigenesis and provided a potential biomarker in breast cancer.

The Quebec Parkinson Network: A Researcher-Patient Matching Platform and Multimodal Biorepository.

BACKGROUND: Genetic, biologic and clinical data suggest that Parkinson's disease (PD) is an umbrella for multiple disorders with clinical and pathological overlap, yet with different underlying mechanisms. To better understand these and to move towards neuroprotective treatment, we have established the Quebec Parkinson Network (QPN), an open-access patient registry, and data and bio-samples repository. OBJECTIVE: To present the QPN and to perform preliminary analysis of the QPN data. METHODS: A total of 1,070 consecutively recruited PD patients were included in the analysis. Demographic and clinical data were analyzed, including comparisons between males and females, PD patients with and without RBD, and stratified analyses comparing early and late-onset PD and different age groups. RESULTS: QPN patients exhibit a male:female ratio of 1.8:1, an average age-at-onset of 58.6 years, an age-at-diagnosis of 60.4 years, and average disease duration of 8.9 years. REM-sleep behavior disorder (RBD) was more common among men, and RBD was associated with other motor and non-motor symptoms including dyskinesia, fluctuations, postural hypotension and hallucinations. Older patients had significantly higher rates of constipation and cognitive impairment, and longer disease duration was associated with higher rates of dyskinesia, fluctuations, freezing of gait, falls, hallucinations and cognitive impairment. Since QPN's creation, over 60 studies and 30 publications have included patients and data from the QPN. CONCLUSIONS: The QPN cohort displays typical PD demographics and clinical features. These data are open-access upon application (http://rpq-qpn.ca/en/), and will soon include genetic, imaging and bio-samples. We encourage clinicians and researchers to perform studies using these resources.

Inducible and coupled expression of the polyomavirus middle T antigen and Cre recombinase in transgenic mice: an in vivo model for synthetic viability in mammary tumour progression.

INTRODUCTION: Effective in vivo models of breast cancer are crucial for studying the development and progression of the disease in humans. We sought to engineer a novel mouse model of polyomavirus middle T antigen (PyV mT)-mediated mammary tumourigenesis in which inducible expression of this well-characterized viral oncoprotein is coupled to Cre recombinase (TetO-PyV mT-IRES-Cre recombinase or MIC). METHODS: MIC mice were crossed to the mouse mammary tumour virus (MMTV)-reverse tetracycline transactivator (rtTA) strain to generate cohorts of virgin females carrying one or both transgenes. Experimental (rtTA/MIC) and control (rtTA or MIC) animals were administered 2 mg/mL doxycycline beginning as early as eight weeks of age and monitored for mammary tumour formation, in parallel with un-induced controls of the same genotypes. RESULTS: Of the rtTA/MIC virgin females studied, 90% developed mammary tumour with complete penetrance to all glands in response to doxycycline and a T50 of seven days post-induction, while induced or un-induced controls remained tumour-free after one year of induction. Histological analyses of rtTA/MIC mammary glands and tumour revealed that lesions followed the canonical stepwise progression of PyV mT tumourigenesis, from hyperplasia to mammary intraepithelial neoplasia/adenoma, carcinoma, and invasive carcinoma that metastasizes to the lung; at each of these stages expression of PyV mT and Cre recombinase transgenes was confirmed. Withdrawal of doxycycline from rtTA/MIC mice with end-stage mammary tumours led to rapid regression, yet animals eventually developed PyV mT-expressing and -non-expressing recurrent masses with varied tumour histopathologies. CONCLUSIONS: We have successfully created a temporally regulated mouse model of PyV mT-mediated mammary tumourigenesis that can be used to study Cre recombinase-mediated genetic changes simultaneously. While maintaining all of the hallmark features of the well-established constitutive MMTV-PyV mT model, the utility of this strain derives from the linking of PyV mT and Cre recombinase transgenes; mammary epithelial cells are thereby forced to couple PyV mT expression with conditional ablation of a given gene. This transgenic mouse model will be an important research tool for identifying synthetic viable genetic events that enable PyV mT tumours to evolve in the absence of a key signaling pathway.

The p110α and p110ß isoforms of PI3K play divergent roles in mammary gland development and tumorigenesis.

Class Ia phosphatidylinositol 3 kinase (PI3K) is required for oncogenic receptor-mediated transformation; however, the individual roles of the two commonly expressed class Ia PI3K isoforms in oncogenic receptor signaling have not been elucidated in vivo. Here, we show that genetic ablation of p110α blocks tumor formation in both polyoma middle T antigen (MT) and HER2/Neu transgenic models of breast cancer. Surprisingly, p110ß ablation results in both increased ductal branching and tumorigenesis. Biochemical analyses suggest a competition model in which the less active p110ß competes with the more active p110α for receptor binding sites, thereby modulating the level of PI3K activity associated with activated receptors. Our findings demonstrate a novel p110ß-based regulatory role in receptor-mediated PI3K activity and identify p110α as an important target for treatment of HER2-positive disease.

PTEN deficiency in a luminal ErbB-2 mouse model results in dramatic acceleration of mammary tumorigenesis and metastasis.

Overexpression and/or amplification of the ErbB-2 oncogene as well as inactivation of the PTEN tumor suppressor are two important genetic events in human breast carcinogenesis. To address the biological impact of conditional inactivation of PTEN on ErbB-2-induced mammary tumorigenesis, we generated a novel transgenic mouse model that utilizes the murine mammary tumor virus (MMTV) promoter to directly couple expression of activated ErbB-2 and Cre recombinase to the same mammary epithelial cell (MMTV-NIC). Disruption of PTEN in the mammary epithelium of the MMTV-NIC model system dramatically accelerated the formation of multifocal and highly metastatic mammary tumors, which exhibited homogenous pathology. PTEN-deficient/NIC-induced tumorigenesis was associated with an increase in angiogenesis. Moreover, inactivation of PTEN in the MMTV-NIC mouse model resulted in hyperactivation of the phosphatidylinositol 3'-kinase/Akt signaling pathway. However, like the parental strain, tumors obtained from PTEN-deficient/NIC mice displayed histopathological and molecular features of the luminal subtype of primary human breast cancer. Taken together, our findings provide important implications in understanding the molecular determinants of mammary tumorigenesis driven by PTEN deficiency and ErbB-2 activation and could provide a valuable tool for testing the efficacy of therapeutic strategies that target these critical signaling pathways.

Tektin 2 is required for central spindle microtubule organization and the completion of cytokinesis.

During anaphase, the nonkinetochore microtubules in the spindle midzone become compacted into the central spindle, a structure which is required to both initiate and complete cytokinesis. We show that Tektin 2 (Tek2) associates with the spindle poles throughout mitosis, organizes the spindle midzone microtubules during anaphase, and assembles into the midbody matrix surrounding the compacted midzone microtubules during cytokinesis. Tek2 small interfering RNA (siRNA) disrupts central spindle organization and proper localization of MKLP1, PRC1, and Aurora B to the midzone and prevents the formation of a midbody matrix. Video microscopy revealed that loss of Tek2 results in binucleate cell formation by aberrant fusion of daughter cells after cytokinesis. Although a myosin II inhibitor, blebbistatin, prevents actin-myosin contractility, the microtubules of the central spindle are compacted. Strikingly, Tek2 siRNA abolishes this actin-myosin-independent midzone microtubule compaction. Thus, Tek2-dependent organization of the central spindle during anaphase is essential for proper midbody formation and the segregation of daughter cells after cytokinesis.