Barbara Nicke

Binding of Ras to Phosphoinositide 3-Kinase p110α Is Required for Ras- Driven Tumorigenesis in Mice

Ras proteins signal through direct interaction with a number of effector enzymes, including type I phosphoinositide (PI) 3-kinases. Although the ability of Ras to control PI 3-kinase has been well established in manipulated cell culture models, evidence for a role of the interaction of endogenous Ras with PI 3-kinase in normal and malignant cell growth in vivo has been lacking. Here we generate mice with mutations in the Pi3kca gene encoding the catalytic p110alpha isoform that block its interaction with Ras. Cells from these mice show proliferative defects and selective disruption of signaling from growth factors to PI 3-kinase. The mice display defective development of the lymphatic vasculature, resulting in perinatal appearance of chylous ascites. Most importantly, they are highly resistant to endogenous Ras oncogene-induced tumorigenesis. The interaction of Ras with p110alpha is thus required in vivo for certain normal growth factor signaling and for Ras-driven tumor formation.

Chromosomal instability determines taxane response

Microtubule-stabilizing (MTS) agents, such as taxanes, are important chemotherapeutics with a poorly understood mechanism of action. We identified a set of genes repressed in multiple cell lines in response to MTS agents and observed that these genes are overexpressed in tumors exhibiting chromosomal instability (CIN). Silencing 22/50 of these genes, many of which are involved in DNA repair, caused cancer cell death, suggesting that these genes are involved in the survival of aneuploid cells. Overexpression of these “CIN-survival” genes is associated with poor outcome in estrogen receptor–positive breast cancer and occurs frequently in basal-like and Her2-positive cases. In diploid cells, but not in chromosomally unstable cells, paclitaxel causes repression of CIN-survival genes, followed by cell death. In the OV01 ovarian cancer clinical trial, a high level of CIN was associated with taxane resistance but carboplatin sensitivity, indicating that CIN may determine MTS response in vivo. Thus, pretherapeutic assessment of CIN may optimize treatment stratification and clinical trial design using these agents.

The Extracellular Matrix Protein TGFBI Induces Microtubule Stabilization and Sensitizes Ovarian Cancers to Paclitaxel

Summary The extracellular matrix (ECM) can induce chemotherapy resistance via AKT-mediated inhibition of apoptosis. Here, we show that loss of the ECM protein TGFBI (transforming growth factor beta induced) is sufficient to induce specific resistance to paclitaxel and mitotic spindle abnormalities in ovarian cancer cells. Paclitaxel-resistant cells treated with recombinant TGFBI protein show integrin-dependent restoration of paclitaxel sensitivity via FAK- and Rho-dependent stabilization of microtubules. Immunohistochemical staining for TGFBI in paclitaxel-treated ovarian cancers from a prospective clinical trial showed that morphological changes of paclitaxel-induced cytotoxicity were restricted to areas of strong expression of TGFBI. These data show that ECM can mediate taxane sensitivity by modulating microtubule stability.

Predictive biomarker discovery through the parallel integration of clinical trial and functional genomics datasets

The European Union multi-disciplinary Personalised RNA interference to Enhance the Delivery of Individualised Cytotoxic and Targeted therapeutics (PREDICT) consortium has recently initiated a framework to accelerate the development of predictive biomarkers of individual patient response to anti-cancer agents. The consortium focuses on the identification of reliable predictive biomarkers to approved agents with anti-angiogenic activity for which no reliable predictive biomarkers exist: sunitinib, a multi-targeted tyrosine kinase inhibitor and everolimus, a mammalian target of rapamycin (mTOR) pathway inhibitor. Through the analysis of tumor tissue derived from pre-operative renal cell carcinoma (RCC) clinical trials, the PREDICT consortium will use established and novel methods to integrate comprehensive tumor-derived genomic data with personalized tumor-derived small hairpin RNA and high-throughput small interfering RNA screens to identify and validate functionally important genomic or transcriptomic predictive biomarkers of individual drug response in patients. PREDICTs approach to predictive biomarker discovery differs from conventional associative learning approaches, which can be susceptible to the detection of chance associations that lead to overestimation of true clinical accuracy. These methods will identify molecular pathways important for survival and growth of RCC cells and particular targets suitable for therapeutic development. Importantly, our results may enable individualized treatment of RCC, reducing ineffective therapy in drug-resistant disease, leading to improved quality of life and higher cost efficiency, which in turn should broaden patient access to beneficial therapeutics, thereby enhancing clinical outcome and cancer survival. The consortium will also establish and consolidate a European network providing the technological and clinical platform for large-scale functional genomic biomarker discovery. Here we review our current understanding of molecular mechanisms driving resistance to anti-angiogenesis agents, the current limitations of laboratory and clinical trial strategies and how the PREDICT consortium will endeavor to identify a new generation of predictive biomarkers.

CERT depletion predicts chemotherapy benefit and mediates cytotoxic and polyploid‐specific cancer cell death through autophagy induction

Chromosomal instability (CIN) has been implicated in multidrug resistance and the silencing of the ceramide transporter, CERT, promotes sensitization to diverse cytotoxics. An improved understanding of mechanisms governing multidrug sensitization might provide insight into pathways contributing to the death of CIN cancer cells. Using an integrative functional genomics approach, we find that CERT‐specific multidrug sensitization is associated with enhanced autophagosome–lysosome flux, resulting from the expression of LAMP2 following CERT silencing in colorectal and HER2+ breast cancer cell lines. Live cell microscopy analysis revealed that CERT depletion induces LAMP2‐dependent death of polyploid cells following exit from mitosis in the presence of paclitaxel. We find that CERT is relatively over‐expressed in HER2+ breast cancer and CERT protein expression acts as an independent prognostic variable and predictor of outcome in adjuvant chemotherapy‐treated patients with primary breast cancer. These data suggest that the induction of LAMP2‐dependent autophagic flux through CERT targeting may provide a rational approach to enhance multidrug sensitization and potentiate the death of polyploid cells following paclitaxel exposure to limit the acquisition of CIN and intra‐tumour heterogeneity. Copyright

Initiation of High Frequency Multi-Drug Resistance Following Kinase Targeting by siRNAs

The sequential use of non-cross resistant cytotoxic agents is the standard of care for advanced solid tumours in order to enhance survival and optimise quality of life. Nevertheless, drug resistance to non cross-resistant agents is commonly witnessed, with clinical response rates to “non cross-resistant” regimens declining as the disease advances. Expression of ABC transporters is unlikely to fully explain this phenomenon, and a clear molecular explanation for this process remains uncertain. A statistical analysis of a recently published RNA interference screen targeting 779 kinases in three cell lines deriving from different tumour types reveals a significant correlation between resistance to paclitaxel and a “non cross-resistant” cytotoxic agent. Furthermore, 20% of kinases that promote resistance to paclitaxel when targeted by RNAi also promote resistance to a non cross-resistant agent within that same cell line, consistent with a tissue-type dependence of multi-drug resistance. Conversely, paclitaxel-specific resistance and sensitising kinases occur less frequently than expected. This indicates that several cell line specific kinases may regulate multi-drug resistance and provide a potential explanation for the phenomenon of drug resistance to non cross-resistant agents witnessed in oncology practice. Furthermore, this work supports efforts to identify common pathways of drug response for future drug discovery programmes.

RNA interference, DNA methylation, and gene silencing: a bright future for cancer therapy?

The gene-silencing process of RNA interference (RNAi) involves the manufacture of short, double-stranded RNA molecules by an enzyme called DICER, which cleaves RNA duplexes into 21–26 base-pair oligomers. These small interfering RNAs (siRNA) cause sequence-specific post-transcriptional gene silencing by guiding an endonuclease, the RNAi-induced silencing complex (RISC), to mRNA. This process has been seen in a wide range of organisms such as Neurospora fungus (quelling), plants (post-transcriptional gene silencing), and mammalian cells (RNAi). Recent reports 1,2

PDE5 inhibition eliminates cancer stem cells via induction of PKA signaling

Cancer stem cells (CSCs) are involved in metastasis and resistance development, thus affecting anticancer therapy efficacy. The underlying pathways required for CSC maintenance and survival are not fully understood and only a limited number of treatment strategies to specifically target CSCs have been identified. To identify novel CSC targeting compounds, we here set-up an aldehyde dehydrogenase (ALDH)-based phenotypic screening system that allows for an automated and standardized identification of CSCs. By staining cancer cells for ALDH activity and applying high-content-based single-cell population analysis, the proportion of a potential CSC subpopulation with significantly higher ALDH activity (ALDHhigh) can be quantified in a heterogeneous cell population. We confirmed high ALDH activity as surrogate marker for the CSC subpopulation in vitro and validated Wnt signaling as an essential factor for the maintenance of CSCs in SUM149 breast cancer cells. In a small molecule screen, we identified phosphodiesterase type 5 (PDE5) inhibition as potential strategy to target CSC maintenance and survival in multiple cancer cell lines. CSC elimination by PDE5 inhibition was not dependent on PKG signaling, and we suggest a novel mechanism in which PDE5 inhibition leads to elevated cGMP levels that stimulate cAMP/PKA signaling to eliminate CSCs.

A Functional Role for CERT in Cancer Drug Induced Autophagy and Prognosis in Her2 Positive Breast Cancer.

CERT was recently identified in a functional genomic siRNA screen for regulators of multi-drug sensitivity. CERT encodes a ceramide transporter which shuttles the pro-apoptotic lipid ceramide from the endoplasmic reticulum to the golgi. Silencing CERT, promoted sensitivity to paclitaxel, doxorubicin, cisplatin and 5-FU in breast, colon and non-small cell lung cancer cell lines. Further analysis of CERT expression in 18 NCI-60 cancer cell lines together with drug sensitivity data for over 5000 drugs within the NCI database, revealed that elevated CERT expression is associated with multi-drug resistance to diverse cancer cytotoxics. CERT mRNA was significantly elevated in HER2 positive breast cancer cell lines. Consistent with these data, in a primary breast cancer tissue microarray cohort of 356 primary breast cancers with outcome data, CERT protein expression was significantly greater in Her2 positive disease (p Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1137.