Cell Shape and Movement
How do Cancer Cells Adopt Different Shapes During Metastasis?
The migration of cells is an essential event during organism development and for many functions in the adult organism. For example immune cells have to migrate to sites of infection in order to eradicate bacteria or viruses. Abnormalities of cell migration are also characteristic of disease processes such as inflammatory diseases or metastatic cancers.
In both normal and pathological settings, cells must reorganize their cell shape in highly dynamic and orchestrated manners in order to migrate. Molecules called Rho-family GTPases act as biochemical switches that couple cytoskeletal organization to distinct environmental signals regulate these shape changes. Rho guanine nucleotide exchange factors (RhoGEFs) and Rho GTPase activating proteins (RhoGAPs) are the principal regulators of Rho-family GTPases.
But the specific signals that activate or inhibit RhoGEFs and RhoGAPs, as well which RhoGTPases are their in vivo targets are largely unknown. Moreover, the downstream effectors of Rho signaling which link Rho activity to specific changes in cell shape (e.g. retraction, or the formations of protrusions and lammelipodia) are poorly understood.
We using are high-throughput combinatorial RNAi screening methods where cell shape is used as a phenotypic readout to identify both upstream regulators and downstream effectors of Rho signaling on a systems-level.
Through previously performed genome scale RNAi screens we have determined the contribution of all RhoGEFs, RhoGAPs, RhoGTPases, kinases, phosphatases, and transcription factors to cell shape. Each gene is assigned a Quantitative Morphological Signature (QMS) which a multi-dimensional vector describing the effects of gene inhibition on morphology.
We are now systematically inhibiting all Rho components in combination with all other, as well as with all known kinases, phosphatases, and transcription factors to determine quantitative genetic interactions that underpin cell shape.
These screens can be used to identify both specific and general regulators of individual Rho components and thus provide novel insight as to how Rho-family GTPase activity is tailored resulting in specific phenotypic output in diverse environmental conditions.
Importantly, performing combinatorial screens with quantitative morphology as a readout requires the development of new computational methods to determine epistastic relationships.
In collaboration with the Berger laboratory (Massachusetts Institute of Technology, Cambridge USA) and the Wong laboratory (Methodist Research Institute, Houston USA) we are continuing to develop these methods and have validated their use in preliminary studies.
What Genes are Responsible for Metastasis?
Complex and highly coordinated changes in morphology occur during cancer cell metastasis. For example, metastatic cancer cells of epithelial origin lose cell-cell contacts and apical-basal polarity, engage once-dormant migratory machinery, remodel the extracellular matrix (ECM) and dynamically regulate integrin-based adhesion.
Importantly, while there are common morphological aspects of metastasis, cells may also alternate between distinct modes of migration, such as mesenchymal versus amoeboid. Understanding how signaling networks that control shape are differentially rewired during oncogenesis is critical for developing safe and effective therapeutics.
Given the recent advances in genomic profiling, we have an unprecedented opportunity to describe the genotype of cancer cells. However, a major challenge in the post-genomic era is to understand which genetic alterations are truly drivers of cancer cell phenotypes. For example, it is not clear which transcriptional changes underpin the ability of metastatic cells to migrate and invade secondary tissues.
A number of studies have identified potential “master” regulators of metastatic phenotypes such as Twist, and RhoC, but the list of downstream effectors that lead to cell shape changes remains far from complete.
One well-characterized example of a link between metastatic cell genotype and phenotype is the switch from E-cadherin to N-cadherin expression that results in a loss of cell-cell contacts and mesenchymal morphology in certain epithelial lines. But we hypothesize there are many more unknown genetic changes that are essential for the morphogenesis of metastatic cells.
We are using methods we have previously developed in order to quantify cell shape in parallel with genome-wide microarray and comparative genomic hybridization techniques to determine how specific, quantitative differences in cell morphology in both 2D and 3D are driven by changes in gene expression and copy number variation.