Fibrolamellar Hepatocellular Carcinoma

An unexpected discovery may pave the way to better treatments for a broad range of cancers. It's been enormously difficult to pinpoint the cause of many cancers -- and many seem to have more than one origin. Fibrolamellar carcinoma (FLC), however, is one that scientists thought they had nailed down. A rare and currently incurable disease that attacks the liver of children, adolescents, and young adults, FLC is caused when a small deletion in chromosome 19 causes the fusion of two genes, a discovery made in 2014 in the lab of Rockefeller's Sanford M. Simon, whose own then-teenage daughter, Elana, had not only been diagnosed with liver disease a few years before, but was a lead on the team that found the fusion. One gene is DNAJB1, which produces heat-shock proteins that encourage cell homeostasis, and the other is PRKACA, the generator of the catalytic subunit of protein kinase A (PKA), which is key to cellular metabolic function. Alteration in kinases, which modify many other molecules, have been implicated in driving many cancers. For the past decade it was thought that this fusion created a Frankenstein-type change in PKA so that it wreaked havoc in the cell. Now researchers in Simon's lab have made a surprising discovery: the fusion protein behaves just like a normal kinase. But cells containing an addition to their catalytic subunit generate the kinase in excessive amounts -- which is the real culprit. "It's actually the overexpression of a protein called PKA that causes the cancer," says first author Mahsa Shirani, a postdoctoral associate in the Laboratory of Cellular Biophysics, headed by Simon. "These findings have the potential to both reveal the pathways of a broad range of cancers and offer new treatment possibilities." The researchers published their results in Cancer Research. A lack of inhibition Shirani's research has been aimed at a deeper understanding of the fused gene's mechanics since her time as a Ph.D. student and teaching assistant in the lab of New Mexico State University biochemist Barbara Lyons, whose research into FLC was driven by her own son's diagnosis with the disease. Like Elana Simon and numerous other patients, her son, Jackson Clark, put his life on hold to research the disease in the Simon lab. His first article from the lab was published last year. Clark passed away from FLC. For the current study, Shirani analyzed tumor tissue samples from FLC patients using mass spectrometry, biochemistry, and immunofluorescence to quantify the level of the protein in patients' tumor tissue. She also compared them to normal liver tissue. Digging deeper, she found that the tumor cells have a molecular imbalance: an increased amount of catalytic proteins exceeds the number of inhibitory ones that normally tamp down and localize the former. This excess has two profound effects on the cell. One is that PKA activity amps up, unchecked. The other is that PKA is now free to move around the cell, wreaking havoc in spots that it usually cannot access, including the nucleus. Shirani's results indicate that the fact that the active catalytic subunit overrules its inhibitory components is what's important, not a structural change in the kinase itself. In testing this theory, the researchers found that they could recreate the disease in human liver cells just by increasing the amount of the normal kinase. They also found that some patients had a completely different gene fused to the front end of the same kinase, PRKACA, indicating that the actual cause of disease couldn't be attributed to the extra piece added to the kinase. "We showed that it doesn't matter what you have fused to the PRKACA gene. It could be DNAJB1 or ATP1B1, or it could be nothing at all -- just PRKACA that has a high protein expression," she says. "Each situation leads to the same phenotype of cancer." The researchers corroborated their findings using a unique tool at their disposal. For the past decade, the Simon Lab has operated the Fibrolamellar Tissue Repository. When the researchers went back through their samples, they came across four patients who had what looked like fibrolamellar but did not have a fusion to PRKACA. Instead, the only alteration they found was a loss of the inhibitory protein, providing more evidence that the amount of catalytic subunit relative to its regulatory components was a key factor in disease formation. Treatment horizon The findings could potentially lead to the first therapeutic treatments for FLC beyond the surgical removal of tumors, says Shirani. (Treatments available to people with common liver cancer are useless for FLC, which has a completely different molecular profile.) One idea is to locate binding sites on the DNAJB1 protein to which a drug inhibitor could bind. Another is tamping down the expression of PKA. The lab is currently investigating both possibilities. The latter approach could have potential beyond FLC, Shirani says, because PKA dysregulation is connected to many other diseases. For example, the adrenal tumor that causes Cushing Syndrome is the result of a mutation in the very same catalytic subunit, PRKACA. (That discovery was made by Rockefeller President Richard P. Lifton in 2014.) As with a potential FLC treatment, the key would be to interfere with signaling processes downstream of PKA's production, before the geyser of protein production disrupts the cell. Shirani also suggests that measuring protein levels produced by mutated genes may be a first step to better understanding any number of cancers: "Perhaps the increased level, or location, of proteins is itself the cause." The findings may also illuminate pathogenesis of disease in general -- one of the many important reasons for investigating rare diseases, which are often viewed as insignificant since they affect so few people, says Simon. "There are so many good reasons to study them," he says. "Many rare diseases are very precisely characterized, making it possible to make rapid progress, and those results can often be generalized to common diseases. For example, we learned about the concept of 'tumor suppressor' from studying the rare childhood cancer retinoblastoma." "I also think that as we more precisely define diseases, we find that many that were thought to be single diseases are actually collections of different rare diseases that share some common characteristic or mechanism," he adds.

New research has shown that cells regulate cAMP/PKA signaling by forming liquid droplets that segregate excess PKA catalytic subunits where they can do no harm. Some cancers may block the formation of liquid droplets, leading to hyperactive signaling and tumor formation. New research by scientists at University of California San Diego has shown that cells regulate cAMP/PKA signaling by forming liquid droplets that segregate excess PKA catalytic subunits where they can do no harm. Some cancers may block the formation of liquid droplets, leading to hyperactive signaling and tumor formation. Healthy cells respond appropriately to changes in their environment. They do this by sensing what's happening outside and relaying a command to the precise biomolecule in the precise domain that can carry out the necessary response. When the message gets to the right domain at the right time, your body stays healthy. When it ends up at the wrong place at the wrong time, you can get diseases such as diabetes or cancer. The routes that messages take inside a cell are called signaling pathways. Cells use only a few signaling pathways to respond simultaneously to hundreds of external signals, so those pathways need to be tightly regulated. New research by scientists at University of California San Diego has uncovered a surprising way that cells regulate signaling pathways. They found that when there are too many messages floating around inside a cell, the messengers form liquid droplets, sequestering themselves away where they can do no harm. The work was recently published in Molecular Cell. "Liquid droplets organize cellular biochemical activities according to spatiotemporal regulation," says Jin Zhang, Ph.D., professor of pharmacology at UC San Diego School of Medicine and senior author on the study. The scientists worked with one of the main routes for cellular communication. It's called the cAMP/PKA signaling pathway for its two main actors -- cAMP (cyclic adenosine monophosphate) and PKA (cAMP-dependent protein kinase). When cAMP receives a signal from the cell's surface, it activates PKA. PKA relays the message to the appropriate domain, whether it's telling a specific gene to produce more protein or stimulating an enzyme to maintain a healthy level of glucose in the blood. It's not that simple, though. PKA carries messages to hundreds of different domains. According to Zhang, "At one moment, PKA needs to be active on the plasma membrane. But the next moment, it needs to come off the plasma membrane and be active on the mitochondrial membrane. Ten minutes later, it really needs to be in the nucleus to turn on transcription." To complicate matters, sometimes cells turn on too much cAMP and PKA. When that occurs, cell signaling becomes hyperactive and indiscriminate. Explains Zhang, "Different microdomains control different things. Let's say you want the cAMP level to be high around calcium channels but low 10 nanometers away. How does the cell achieve that? By controlling cAMP." But, she goes on, "PKA is the same. Typically, it's recruited to specific domains by anchoring proteins. But if the PKA activity is too high, it will activate domains it's not supposed to be activating. That's loss of specificity." That's why, according to the new research, cells form liquid droplets to ensure that the right message gets to the right domain at the right time. When the scientists analyzed the composition of the liquid droplets, as well as the timing of when they were formed, they found that cells formed the droplets using a subunit of PKA when too much cAMP and PKA were being turned on. In that way, the droplets sequestered the excess cAMP and PKA and tamped down non-specific signaling. In previous work, the authors found that a rare type of liver cancer called Fibrolamellar Carcinoma (FLC) blocks formation of these liquid droplets, resulting in uncontrolled cell signaling. "We think that disappearance of the liquid droplets is one major contributor to this hyperactive signaling that leads to tumorigenesis," said Zhang. FLC is a rare but devastating disease. It typically affects people under the age of 40 with healthy livers. The authors of this paper are hoping to investigate whether other cancers also cause a loss of liquid droplets and what the molecular mechanisms are behind it. Their ultimate goal is to design a molecular therapeutic to treat FLC -- "anything," says Zhang, "that helps us address the unmet needs of FLC patients." The authors of this study include Julia C. Hardy, Emily H. Pool, Jessica G.H. Bruystens, Xin Zhou, Qingrong Li, Daojia R. Zhou, Max Palay, Gerald Tan, Lisa Chen, Jaclyn L.C. Choi, Ha Neul Lee Dong Wang, Susan S. Taylor, Sohum Mehta, Jin Zhang at University of California San Diego and Stefan Strack at University of Iowa. This work was supported by the National Institutes of Health and Fibrolamellar Cancer Foundation.

Researchers have translated a cutting-edge tactic for treating genetic disorders into a fresh approach for potentially stymying cancer. Cell division is the generative spark of nearly every lifeform on Earth. But if healthy growth goes unchecked, cell division can turn lethal, overwhelming the organism. Such is the case with so-called oncogenes. When altered by a mutation, these growth-moderating genes go haywire, producing a geyser of cancer cells as a result. Oncogenes are also insidiously adept at generating tumors that over time become genetically "independent" from their origin, so it has been exceedingly difficult for scientists to shut down their replication at the source. Now Rockefeller University researchers have discovered a new way to target the oncogene behind a rare and often deadly liver disease using small interfering RNAs (siRNAs), an innate mechanism for silencing gene expression. As described in Molecular Therapy, the researchers slipped siRNAs inside fibrolamellar hepatocellular carcinoma (FLC) cells through a surface receptor, where they blocked the oncogene from producing illness-inducing proteins that lead to tumor formation. It's the first time that siRNAs have been used to hobble the progression of FLC, says first author Christoph Neumayer, a Ph.D. student in Rockefeller's Laboratory of Cellular Biophysics, headed by Sanford M. Simon. But the findings have wider applications as well. "That's the really exciting part," says Neumayer. "This is proof of concept that siRNAs can be used for FLC, as well as adult liver cancers, which are much more common, and other tumor types elsewhere in the body." The fusion oncogene FLC is caused by the fusion of two genes located on chromosome 19: DNAJB1, which produces heat-shock proteins that encourage cell homeostasis, and PRKACA, which produces an enzyme called kinase A that's key to cellular metabolic function. The resulting fusion, known as DNAJB1::PRKACA, brings together the proteins and a catalytic subunit of the kinase. This dysfunctional union promotes FLC formation, but how, exactly, is still unknown. Researchers in Simon's lab discovered that this fusion caused FLC in 2014, several years after Simon's own teenage daughter, Elana, was diagnosed with liver disease. After having the resulting tumor removed -- the only treatment option available to people with FLC -- Elana went on to collaborate with her father to unearth its cause, publishing the results in Science. Since then, Simon and his group have continued to reveal the mechanisms behind the disease as well as develop therapeutics. The siRNA research is part of a three-part strategy which also includes repurposing drugs for treating FLC and using molecules known as PROTACs to selectively degrade the DNAJB1::PRKACA protein that drives FLC. The drug repurposing is in advanced preparation for a clinical trial, and some of the PROTAC work is part of an international collaboration that was just awarded a $25 million Cancer Grand Challenges grant to develop treatments for high-risk, oncogene-driven cancers in children. Recently, Simon's lab discovered that they could completely stop FLC tumor growth in mice using shRNAs, short sequences of RNA that can be engineered to disrupt mRNA. The approach also caused many tumors to shrink or disappear. Having shown that quelling the fused oncogene killed tumor cells, they decided to experiment with derailing kinase A activity, which research indicated was driving tumor growth. Unlocking the cell door The problem was, fused kinase A and its "wild type" form are nearly identical, so "any drug you developed that blocks fused kinase A activity would affect all kinase A, including in normal cells," Neumayer says. "In other words, you would incur a host of problematic side effects." They had to find a way to disable toxic kinase while containing the impact so that kinase in other cells weren't affected. To do that, they specifically designed siRNAs to aim at the fusion. But because siRNAs cannot cross cell membranes, the researchers needed to figure out a way to get the siRNA inside. Luckily, a door into the cell had already been identified: a receptor called ASGR1 that's only expressed in liver cells. Its key had been too: a binding molecule known as GalNAc. Together, the duo function as a waste disposal team. GalNAc conjugate therapies, which attach siRNA molecules to the ligand to deliver therapies inside cells, are already on the market for conditions such as hereditary transthyretin-mediated amyloidosis and atherosclerotic cardiovascular disease. The team wondered if they could use the same approach to thwart the kinase driving FLC tumors. Stabilization and shrinkage To test their theory, the researchers attached a custom siRNA to the receptor's ligand and studied the method in a variety of tumor models in mice and in human cell cultures. The conjugate therapy was not only successfully delivered into the cells in all contexts, but it also caused a reduction of mRNA in the oncogene, preventing the production of the protein and resulting in the tumors stagnating or shrinking, unable to grow. They also did not detect any liver toxicity in the mice, indicating the animals tolerated the siRNAs well. "What we saw was tumor inhibition, when we had hoped to outright kill the tumor," Neumayer notes. "Our future direction will be to try to figure out how to improve that." They also tested the specificity of the siRNA by injecting it into tumor cells of another type of liver cancer. It had no toxic impact on them -- exactly as the researchers hoped. "We wanted to show that our siRNA is so targeted to the FLC fusion oncogene that it has no side effects on other cells -- even on other cancer cells," he notes. Neumayer says the findings suggest that siRNAs may be effective beyond FLC, capable of treating tumors throughout the body with a high degree of specificity: "I think siRNA medicines as a class will have a big impact over time as a new kind of genetic therapy."