Genetic Engineering & Biotechnology News

DEC 2017

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8 | DECEMBER 2017 | GENengnews.com | Genetic Engineering & Biotechnology News Turning Cell Signaling against Cancer See Cell Signaling on page 10 in this pathway has been linked to changes in oncogenes and tumor-suppressor genes. Studies in recent years have also revealed that this pathway is involved in regulating the epigenome. Some of the challenges in identifying compounds that modulate the PI3K-Akt pathway are that its regulation is complex, multiple regulatory mechanisms are involved, and many of them cross-talk with other signaling pathways. An- other challenge stems from the fact that even though very tumor-specific Akt activators have been well-characterized as potential drug targets, specific inhibitors and possible com- bined treatment options have yet to be developed. "We would like to determine whether we could find can- cer-specific upstream regulators of Akt that are not present or functional in normal cells," informs Dr. Liu. In Dr. Liu's laboratory, investigators are using multidisci- plinary experimental approaches to explore signaling events mediated by protein modifications and protein-protein inter- actions that are dysregulated under pathological conditions in the PI3K-Akt pathway. The investigators hope to identify enzymatic inhibitors and discover antibodies that could be used as therapeutic tools. "By better understanding the interplay between key can- cer signaling events, we would like to connect cancer signal- ing, metabolism, and epigenetics," states Dr. Liu. "Our ulti- mate goal is to identify and develop new targeted therapies to combat cancer." Single-Molecule Studies Cancer research and therapeutic development is increas- ingly focused on tumor heterogeneity, or cell-to-cell vari- ability in genetic, epigenetic, morphological, and phenotypic profiles. Historically, one of the main sources of cell-to-cell variability has been protein expression variation. "Protein expression variation certainly is part of heteroge- neity, but our data argue that molecular processes themselves also contribute to heterogeneity," says Jay. T. Groves, Ph.D., professor of chemistry at the University of California, Berke- ley. "These processes are inherently stochastic." The take-home message from his team's work, Dr. Groves indicates, is that certain molecular processes fluc- tuate between long-lived functional states. These fluctua- tions, he adds, likely "contribute to cell-to-cell heteroge- neous behaviors." In a proof-of-concept study, Dr. Groves and colleagues examined the activation of H-Ras, a small guanosine tri- phosphatase (GTPase), by Son of Sevenless (SOS), a guanine nucleotide exchange factor (GEF). 1 Dr. Groves' team used a single-molecule enzymatic assay to assess the activation of membrane-bound GTPases by GEFs. This approach revealed that SOS undergoes stochastic fluctuations between distinct, long-lived functional states. These states may last more than 100 seconds, reaching time scales characteristic of receptor signaling processes. Historically, studies have examined cellular populations, and parameters have been expressed in the form of mean val- ues, "averaging out" fluctuations between different activity states. Hidden within ensemble averages, these fluctuations eluded capture. "We use our membrane microarray technology to ob- serve H-Ras activation by SOS molecules, assessing large numbers of molecules while still tracking each molecule individually," details Dr. Groves. "In this way, it becomes experimentally practical to map out the whole distribution. We don't have to rely on average measurements—that will be very important." In studies of live cells, certain kinds of variability in pro- tein characteristics tend to remain obscure. For example, proteins in different subcellular locations may have different post-translational modifications. Also, cellular distributions of post-translational modifications may be heterogeneous. Both these sorts of variability may be lost during purification. "One of the technical challenges is how to study molecules that are unpurified, in their native state," advises Dr. Groves. Other proteins have been relatively resistant to structure- function analyses because of challenges in reconstituting them in vitro. SOS is one such protein. It has a disordered C-terminal proline-rich domain that makes purification and X-ray crystallography difficult. Several studies have linked truncations in the proline-rich domain of SOS to human cancers. 2,3,4 To interrogate the struc- ture-function relationship and dynamics of full-length native SOS, Dr. Groves and colleagues developed a membrane micro- array assay that uses single SOS molecules from raw cell lysates. "In our study, the cell lysates still originate from many cells," notes Dr. Groves. "One of the challenges will be to do an experiment on the cytoplasm from a single cell, so that we get to see what one cell is doing rather than looking at averages." Despite relying on averages, the study revealed that the proline-rich domain suppresses SOS by reducing the kinetic rate of activation. The process occurred only on the mem- brane surface, in an activity that was independent of the N- terminal domain of the molecule. "As technologies get better," predicts Dr. Groves, "we will see more of these large- scale single-molecule studies." Splice-Switching Approaches A new approach to fighting prostate cancer combines the actions of steric-blocking splice-switching oligonucleotides (SSOs) and a novel nanotechnology-based approach for tar- geted delivery of DNA to tumor cells. "In our nanotechnol- ogy-based approach, we use specific SSOs that change their conformation under acidic conditions," says Alexander V. Kazansky, Ph.D., associate professor in health and biomedi- cal science at The University of Texas, Rio Grande Valley. Drug Discovery Feature Continued from page 1 The PI3K/mTOR/Akt signaling pathway plays essential roles in cell physiology, and hyperactivation of this signaling contributes to tumorigenesis. Identification of cancer- specific regulatory mechanisms for this pathway will shed new light on targeted therapies to treat cancers with Akt hyperactivation.

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