Carolyn H. Michnoff

Synthetic lethal screen identification of chemosensitizer loci in cancer cells

Abundant evidence suggests that a unifying principle governing the molecular pathology of cancer is the co-dependent aberrant regulation of core machinery driving proliferation and suppressing apoptosis. Anomalous proteins engaged in support of this tumorigenic regulatory environment most probably represent optimal intervention targets in a heterogeneous population of cancer cells. The advent of RNA-mediated interference (RNAi)-based functional genomics provides the opportunity to derive unbiased comprehensive collections of validated gene targets supporting critical biological systems outside the framework of preconceived notions of mechanistic relationships. We have combined a high-throughput cell-based one-well/one-gene screening platform with a genome-wide synthetic library of chemically synthesized small interfering RNAs for systematic interrogation of the molecular underpinnings of cancer cell chemoresponsiveness. NCI-H1155, a human non-small-cell lung cancer line, was employed in a paclitaxel-dependent synthetic lethal screen designed to identify gene targets that specifically reduce cell viability in the presence of otherwise sublethal concentrations of paclitaxel. Using a stringent objective statistical algorithm to reduce false discovery rates below 5%, we isolated a panel of 87 genes that represent major focal points of the autonomous response of cancer cells to the abrogation of microtubule dynamics. Here we show that several of these targets sensitize lung cancer cells to paclitaxel concentrations 1,000-fold lower than otherwise required for a significant response, and we identify mechanistic relationships between cancer-associated aberrant gene expression programmes and the basic cellular machinery required for robust mitotic progression.

A genome-wide RNAi screen for Wnt/β-catenin pathway components identifies unexpected roles for TCF transcription factors in cancer

The Wnt family of secreted proteins coordinate cell fate decision-making in a broad range of developmental and homeostatic contexts. Corruption of Wnt signal transduction pathways frequently results in degenerative diseases and cancer. We have used an iterative genome-wide screening strategy that employs multiple nonredundant RNAi reagents to identify mammalian genes that participate in Wnt/β-catenin pathway response. Among the genes that were assigned high confidence scores are two members of the TCF/LEF family of DNA-binding proteins that control the transcriptional output of the pathway. Surprisingly, we found that the presumed cancer-promoting gene TCF7L2 functions instead as a transcriptional repressor that restricts colorectal cancer (CRC) cell growth. Mutations in TCF7L2 identified from cancer genome sequencing efforts abolish its ability to function as a transcriptional regulator and result in increased CRC cell growth. We describe a growth-promoting transcriptional program that is likely activated in CRC tumors with compromised TCF7L2 function. Taken together, the results from our screen and studies focused on members of the TCF/LEF gene family refine our understanding of how aberrant Wnt pathway activation sustains CRC growth.

PAS kinase: An evolutionarily conserved PAS domain-regulated serine/threonine kinase

PAS domains regulate the function of many intracellular signaling pathways in response to both extrinsic and intrinsic stimuli. PAS domain-regulated histidine kinases are common in prokaryotes and control a wide range of fundamental physiological processes. Similarly regulated kinases are rare in eukaryotes and are to date completely absent in mammals. PAS kinase (PASK) is an evolutionarily conserved gene product present in yeast, flies, and mammals. The amino acid sequence of PASK specifies two PAS domains followed by a canonical serine/threonine kinase domain, indicating that it might represent the first mammalian PAS-regulated protein kinase. We present evidence that the activity of PASK is regulated by two mechanisms. Autophosphorylation at two threonine residues located within the activation loop significantly increases catalytic activity. We further demonstrate that the N-terminal PAS domain is a cis regulator of PASK catalytic activity. When the PAS domain-containing region is removed, enzyme activity is significantly increased, and supplementation of the purified PAS-A domain in trans selectively inhibits PASK catalytic activity. These studies define a eukaryotic signaling pathway suitable for studies of PAS domains in a purified in vitro setting.

Second messenger effects on the myosin phosphorylation system in smooth muscle.

It has been shown that with initial and tonic contractions stimulated by a cholinergic muscarinic agonist in tracheal smooth muscle, the extent of myosin heavy chain phosphorylation remains low. Cholinergic stimulation of tracheal smooth muscle results in formation of both monophosphorylated and diphosphorylated myosin light chain, although the amount of diphosphorylated light chain is substantially less than monophosphorylated light chain. Phosphorylation of the single serine site on myosin light chain by myosin light chain kinase is the primary phosphorylation that is associated with activation of smooth muscle contraction. A general scheme for the physiological regulation of smooth muscle contractility can be proposed. Upon activation of cell surface receptors by neurotransmitters or hormones, phosphoinositide metabolism is stimulated to form InsP3. The InsP3 rapidly releases Ca2+ from sarcoplasmic reticulum which then binds to calmodulin. The Ca2+/calmodulin complex binds to myosin light chain kinase which then phosphorylates myosin light chain. It appears that the rate of conversion of myosin light chain kinase from an inactive to an active enzyme may be a significant rate limiting step for the initiation of myosin light chain phosphorylation. Once the kinase is activated, phosphorylation of myosin light chain may reach maximal values within 2 s with neural stimulation, followed by the slower rate of force development. Protein kinase C does not phosphorylate myosin light chain or heavy chain during the initial or tonic phases of contraction. However, this general scheme for smooth muscle contraction does not exclude the possibility of other regulatory processes involved in sustained contractions.