siRNA knockdowns turn up intriguing HER2 targets
At best, only 20 to 30 percent of women with HER2-positive breast cancer respond to trastuzumab. That grim reality has prompted the Breast Cancer Program to search for new drug targets that, when inhibited, might synergize with trastuzumab to yield better outcomes during initial treatment. In one SPORE project, Randy King, MD, PhD, associate professor of cell biology at HMS, is using siRNA technology to do just that. Rather than looking for all possible needles in the genomic haystack, however, King is aiming at known cancer culprits — kinase genes — whose products lend themselves to small-molecule inhibition and thus more rapid translation into therapies.
In collaboration with Ben Neel, former member of the DF/HCC Breast Cancer Program and current director of the Ontario Cancer Institute, King is hunting for kinase genes that when knocked down by siRNAs prevent the proliferation or survival of HER2-overexpressing cells. The project may also uncover new insights about the biology of HER2-associated signaling pathways.
In the primary screen, King and colleagues muted expression of approximately 600 kinase (and some phosphatase) genes to see which ones affect growth in HER2+ cell lines; in parallel, they conducted the same assays in the presence of trastuzumab. In vitro, trastuzumab inhibits cell proliferation by about 40 percent, giving investigators a handy yardstick by which to measure additional growth inhibition by siRNA knockdown. Although one objective of this work is to identify genes that induce cell death in the presence of trastuzumab but not in its absence, the screen has also turned up genes that inhibit proliferation of HER2-amplified cells independent of trastuzumab. The 100 most effective genes found in the first screen are now being validated in secondary screens in a variety of HER2+ cell lines to confirm their selectivity for HER2-amplified cells. Those validated are then tested across a panel of other breast cancer cell lines – including HER2-negative. “We’re characterizing the results from these kinase screens to understand whether we’ve found any genes specific for HER2-overexpressing cells,” says King. “And we’re still wrestling with that question.”
Validation: the hard work ahead
The real challenge, he says, is winnowing down genes to a subset that are truly selective to the HER2 pathway and physiologically relevant. “We’re being very careful in confirming that the effects of a given siRNA are indeed due to a knockdown of the intended target, as opposed to the so-called “off-target phenomenon.” Because siRNAs are only 21 nucleotides long, they can occasionally hit the same sequences on other genes. To lower that likelihood, King is using four different siRNAs in the secondary screens, each one aimed at a distinct region of the gene of interest. “If we find that all four siRNAs give us the same phenotype, it’s much more likely the consequence of knockdown of the intended gene than of off-target effects.”
The second round of screening also provides a more information-rich readout, measuring both cell number and metabolic activity simultaneously, thus allowing investigators to correlate the two. Moreover, King plans to use time-lapse microscopy to characterize how cells respond when a particular kinase gene is knocked down. “Sometimes cells round up and die right away, or flatten out and just sit there,” says King, who uses time lapse routinely in his other research. “Observing cells over time gives us more insight into the potential mechanisms by which loss of gene function might be inhibiting proliferation.”
Will future avenues of investigation screen kinase genes in trastuzumab-resistant cell lines? Or in combination with lapatinib? “These are all interesting questions,” acknowledges King. “But validating these genes is where the hard work lies right now.”