PRKACA

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.

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."

体外培养类器官的想法和尝试由来已久，这一目标在2009年发生了重要的突破。荷兰的科学家Hans Clevers 博士是肠道干细胞生物学的先驱。他是第一个发现干细胞特异性标记物 (Lgr5) 的人，他的实验室用它来分离能够再生肠、结肠和其他器官的肠道干细胞。2009年，他在《Nature》杂志上发表文章”Single Lgr5 stemcells build crypt villus structures in vitro without a mesenchymal niche“，他展示了人类肠道干细胞可用于产生小肠、胃和肝脏作为可移植的类器官。他的工作对从器官功能到癌症的广泛生物学问题产生了深远的影响。2017年，Clevers教授研究组完成了规模宏大的乳腺癌类器官项目，建立全球首个乳腺癌类器官库。近日，他的研究团队对人类肝细胞类器官进行了CRISPR工程改造，以重建不同的纤维板层癌 (FLC)背景，包括主要的遗传改变、DNAJB1 -PRKACA融合，以及最近报道的FLC 样肿瘤背景，包括BAP1和PRKAR2A的失活突变。这些工程化的人体类器官模型有助于 FLC 的研究。该研究题为“Organoid models of fibrolamellar carcinoma mutations reveal hepatocyte transdifferentiation through cooperative BAP1 and PRKAR2A loss”，发表在Nature communications杂志。来源：https://doi-org.libproxy1.nus.edu.sg/10.1038/s41467-023-37951-6纤维板层癌 (FLC) 是一种罕见的致命性原发性肝癌,主要影响青少年和年轻人。FLC 的有效疗法难以捉摸，因为 FLC 肿瘤很少对批准用于肝细胞癌 (HCC) 的化疗和靶向疗法产生反应，使患者存活率很低。为了改变这一点，非常需要新的治疗策略。新型人体类器官模型该项目的研究组组长 Benedetta Artegiani 教授利用创新技术共同领导了这项关于纤维板层癌的新研究。这使研究人员能够更好地了解 FLC 中发现的不同突变的生物学后果，并研究肿瘤的生物学。需要这些新的研究策略来了解肿瘤产生的原因，并确定可能的目标以更好地治疗该疾病。作者说：“我们在研究中使用了健康的人类肝脏类器官。在这里，我们开发了一系列这些微型器官，它们都具有不同的 DNA 变化和突变，这些变化和突变以前与 FLC 有关。我们使用“分子剪刀”的 DNA 修饰技术 CRISPR-Cas9 改变了类器官的遗传背景。由于其稀有性，可供研究的肿瘤组织并不多。但因为这项技术，我们现在能够研究这种肿瘤类型。”不同的基因突变导致不同程度的攻击性研究人员通过使用 CRISPR-Cas9 修饰蛋白激酶 A (PKA) 复合体构建了肝脏类器官模型。PKA 是一种复杂的信号蛋白，能够打开或关闭其他蛋白质。这种“蛋白质开关”由不同的单元组成，每个单元由不同的基因编码。通过突变改变不同单位的功能对 FLC 的发作至关重要。这些类器官含有所谓的突变融合基因 DNAJB1-PRKACA。这种 DNA 变化在 FLC 肿瘤中很常见。作者说：“在类器官中重建这种突变时，我们发现它确实能够反映我们在 FLC 患者身上看到的肿瘤的多种特征。然而，这种单一突变对肝细胞的整体细胞和分子行为造成了相当轻微的影响。”当他们引入另一组 DNA 变化时，情况完全改变，这种变化也在 FLC 患者中发现。第二个背景不仅包含 PKA 基因之一 PRKAR2A 的突变，而且还包含另一个名为 BAP1 的基因。在这种情况下，类器官呈现出侵袭性癌症的典型特征。这表明不同的遗传 FLC 背景导致不同程度的肿瘤侵袭性。除此之外，由 BAP1 和 PRKAR2A 突变引起的转化效应大大增强，使细胞能够适应不同的环境。这可能解释了 FLC 肿瘤形成过程中细胞不受控制的生长。来源：https://doi-org.libproxy1.nus.edu.sg/10.1038/s41467-023-37951-6研究人员得出结论，尽管 PKA 基因的突变至关重要，但它们可能不足以促进 FLC 的发展。这些发现开启了寻找 FLC 肿瘤中与 PKA 突变一起发生的其他因素的可能性。这也可能被潜在地用于未来可能的治疗该形式的儿童癌症的方法。揭示纤维板层癌的起源细胞为了能够开发新疗法，了解癌症本身的生物学特性也很重要。第一步是了解癌症起源于哪种细胞类型：细胞源。了解特定基因缺陷在 FLC 启动和原始细胞中的重要性对于了解肿瘤随后的行为可能至关重要。然而，在研究过程中，研究人员发现这对 FLC 来说特别困难。作者认为，主要原因是这些肿瘤具有肝细胞和导管细胞的特征，这是肝脏中最重要的两种细胞。作者的类器官结果表明，PRKAR2A 和 BAP1 的共同作用将原本健康的肝细胞转化为导管细胞，并增加了癌症干细胞的特征。这种将一种细胞类型转化为另一种细胞类型的过程称为转分化。这是一个特别有趣的现象，可能发生在各种肿瘤中，并且使得细胞来源的鉴定变得非常困难。然而，通过使用作者构建的类器官模型，我们能够发现肝细胞是可能的细胞来源。来源：https://doi-org.libproxy1.nus.edu.sg/10.1038/s41467-023-37951-6总之，这项研究极大地促进了对 FLC 的理解，并为进一步研究如何更好地治疗这种罕见癌症类型奠定了研究基础。对遗传缺陷的见解可能会为患有这种疾病的儿童带来新的治疗方法。了解特定基因缺陷在 FLC 启动中的重要性在未来也可能有助于更好地了解患者之间的肿瘤异质性和反应。来源：https://www-nature-com.libproxy1.nus.edu.sg/articles/s41467-023-37951-6#Sec2撰文 |Cathy编辑 | cici精彩推荐：1、难怪喝咖啡能瘦！Nutrients：贝勒医学院最新研究每天喝两杯以上咖啡，可有效改善肠道有益菌丰度！2、告别染发剂？Nature重磅：科学家发现白发可逆变黑的“开关”！3、颠覆现有理论！Cell子刊：原来神经元也吃糖，改善大脑糖代谢或可治疗老年痴呆，保持大脑健康！4、举铁吧，头脑还能更聪明！Neuroscience新发现 ：锻炼肌肉有利于增强海马体功能，让大脑更健康！5、PNAS：14万人数据显示，频繁食用油炸食品，焦虑和抑郁风险分别增加 12% 和 7%！