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All Cancers May Fall Into One of Two Classes

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In research published in the journal Cancer Cell, scientists have divided all cancers into two groups, based on the presence or absence of a protein called the yes-associated protein (YAP). Dr. Rod Bremner, senior scientist at the Lunenfeld-Tanenbaum Research Institute, and fellow researchers set out to investigate why only certain cancers show high-frequency loss of a tumor suppressor gene called RB and in the process they discovered that cancers can be divided into two classes, known as YAPon and YAPoff. These classes have varied characteristics and respond differently to therapeutics.

Technology Networks
spoke with Bremner to find out more about this cancer classification technique and the possible implications for future cancer treatments.

Kate Robinson (KR): What is the function of YAP?

Rod Bremner (RB):
YAP and its cousin, WWTR1, better known as TAZ (transcriptional coactivator with PDZ-binding motif), regulate tissue size during fetal development. YAP switches on genes that promote cell division and cell survival, increasing tissue size. Enzymes that keep YAP in check are called the “Hippo” kinases. The battle between Hippo kinases and YAP was first discovered in fruit flies (Drosophila). The fly equivalent of YAP is “Yorkie”. Loss of the red light (Hippo) activates the green light (Yorkie/YAP), causing massive tissue overgrowth. The fruit-fly-field often names a gene based what happens when it is deleted. Hence “Hippo” kinases. The well-studied pro-cancer role of YAP fits this classic pro-division and pro-survival function of this protein. Our work highlights contexts in which YAP does not follow this rule, but instead has the opposite effect and stops cell growth. The cancers in which YAP is an anti-cancer protein include a large group of neuroendocrine and neural cancers, as well as essentially all blood cancers.

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KR: Do cancer cells display different characteristics depending on if they are YAPon or YAPoff?

RB: The first solid cancer we found to be YAPoff was the rare childhood eye cancer, retinoblastoma. The second one was the deadly adult cancer, common in smokers, small cell lung cancer (SCLC). We then performed a pan-cancer assessment and found numerous neural and neuroendocrine cancers that are YAPoff, as well as all types of leukemia and lymphoma. YAPon cancer represents all the other solid tumors, including adenocarcinomas, sarcomas and others. 

There are several major differences between YAPoff and YAPon cancers.

Stickiness: In culture, YAPoff cancers float in the media or adhere very weakly to the culture dish.  In contrast, YAPon cancers stick firmly and must be removed enzymatically. We found that YAP explains this contrary behavior. Thus YAP induces multiple genes that promote adhesion of cells to extra-cellular matrix. When YAP is off, these genes are off, so cells don’t adhere. When we force YAP to be on in YAPoff cancers, it activates these genes and cells now adhere to the dish. Adhesion affects many cell properties, such as migration, survival and responsiveness to drugs, so it can dramatically alter how cancers behave. 

Drug and gene sensitivities: We found that YAPoff cancers are more sensitive to certain drugs than YAPon cancers, and vice versa. This also applies to the gene dependencies of either class. Genes that are essential for survival in YAPoff cancers are dispensable in YAPon cancers, and vice versa. Moreover, the gene dependencies and drug sensitivities overlap e.g., YAPoff cancers are sensitive to drugs that inhibit a protein called NAMPT, and the top ranked selective dependent gene in YAPoff cancers is NAMPT. This insight could be helpful in designing improved therapies for YAPoff cancers.

RB gene loss: We didn’t set out to deduce a pan-cancer rule. Rather, we wanted to understand why only certain cancers show high frequency loss of a tumor suppressor gene called RB. We found that RB gene inactivation is far more frequent in solid YAPoff cancers versus other cancers. So there is a correlation between cells that hate YAP and the ability to exploit RB loss to promote tumor growth. Both retinoblastoma and SCLC actually originate from cell types that have undetectable levels of YAP/TAZ, which helps explain why these cells are prone to tumor growth upon RB loss. Moreover, cancers can jump from the YAPon to YAPoff state to evade therapy, and that often co-occurs with RB gene inactivation. Thus, RB loss and YAP/TAZ-absence often go hand in hand.

KR: Does this study offer any insight into why treatments for cancers are not always successful

Certain cancers, for example adenocarcinomas of the lung and prostate, respond well to new drugs that target proteins essential for their growth and survival. Typically, however, drug-resistant variants of the cancer cells arise. Drug resistance can be achieved by a big change in cell type. Thus, instead of being epithelial in nature, adenocarcinomas can become neuroendocrine, a very different type of cell. This big change allows them to switch to a different circuitry to grow and survive, so the drugs that worked before are no longer effective. We showed that the jump from prostate or lung adenocarcinoma to neuroendocrine cancer represents a switch from the YAPon to the YAPoff state. Thus, cancers can actually jump binary superclasses to evade therapy.

KR: What experimental methods did you use in the Cancer Cell study, and why?

Genetics. We performed many genetic experiments to either remove YAP/TAZ (“Loss of function”, LOF), or to increase YAP/TAZ levels (“Gain of function”, GOF). LOF studies showed that YAP/TAZ are critical for the growth of YAPon but not YAPoff cancers. GOF studies show that forced expression of YAP or TAZ causes YAPoff cancers to stop growing. LOF and GOF studies were performed in vivo in murine retina and lung, and in vitro in multiple human cancer cell lines from multiple tissues, representing many years of effort. We also mined DepMap, a concerted effort to study the degree to which each gene is essential in hundreds of cancer cell lines. This analysis confirmed that YAP or TAZ deletion does not affect YAPoff cancers, but inhibits YAPon cancers. Even in YAPon cases where deleting YAP or TAZ had little effect, we showed that deleting both blocked growth. Thus, essentially all YAPon cancers need YAP/TAZ, and all YAPoff cancers hate them.

Transcriptomics. Human cells have ~20,000 genes, and each cell type has its own “transcriptome” (list of genes that are active). After finding that YAP/TAZ suppress retinoblastoma and SCLC, we explored the transcriptomes of hundreds of cancers to find other YAPoff types. Principal Component Analysis (PCA) finds genes that show the greatest difference in levels across cancers. The top ranked genes (i.e., wildly different across the entire cancer spectrum) included YAP, TAZ and a set of genes that control adhesive behavior, such as Integrins. These genes were off/low in YAPoff cancers, and on/high in YAPon cancers. Discovery of this gene set (unimaginatively termed “PC1+ in our paper) was a defining moment, as it revealed the binary pan-cancer classes for the first time.

By combining GOF studies with transcriptomics we showed that YAP reactivates the PC1+ adhesion genes in YAPoff cells. This result contrasts YAP function in YAPon cancers, where it is well-known to induce genes that promote cell division.  Extensive work then showed that YAP achieves both these contrasting outcomes through binding to TEAD, a family of DNA binding proteins. TEAD targets YAP to the genes that it activates. We studied the TEAD targets and found that they bind different genes in YAPoff vs. YAPon cells. Data mining further revealed that YAP/TEAD cooperate with different proteins in YAPon vs. YAPon cancers. These molecular data explain why YAP has such contrasting effects the binary classes.

Drug sensitivity. To ask whether the binary classes display unique drug sensitivity profiles we mined the Cancer Therapeutics Response Portal (CTRP) and the Genomics of Drug sensitivity in Cancer (GDSC) databases. These team projects are working through hundreds of drugs and cancer cell lines to identify how effective each drug is against different cancers. Devising a weighted sensitivity score, we found several drugs that are better at inhibiting YAPoff vs. YAPon cancers, and vice versa. We further showed that YAP directly influences these differences. Mining the DepMap database, we found that the genes that are selectively essential in YAPoff cancers mirror the drug sensitivities. For example, multiple NAMPT inhibitors were hits and, remarkably, NAMPT was the top ranked gene selectively essential in YAPoff but not YAPon cancers. Another gene that operates in this pathway was also a high ranking hit. Thus, genetic and pharmaceutical approaches identified the same vulnerabilities in YAPoff cancers.

Functional Genomics. A CRISPR screen of YAP-induced and/or YAP-correlated targets revealed that YAP induces an Integrin complex that drives growth arrest in YAPoff cancers. Peptide inhibition studies confirmed this hit from the functional genomics screen. Thus, these adhesion genes are down-regulated because otherwise, they block the growth of YAPoff cancers.

KR: What implications do you envision the current study will have on the development of novel cancer therapeutics?

One of the greatest problems in treating cancer is its complexity. There are so many mutations that promote tumor growth, and each tumor can contain multiple clones with different sets of mutations. Such variation is a challenge for precision medicine, which seeks to identify each druggable mutation and target them to inhibit tumor growth. A separate approach is to uncover overarching cancer properties, and thus to identify broadly applicable vulnerabilities. Our work simplifies cancer into just two types and identifies therapies that are more effective in either class.

A second huge problem in treating cancer is plasticity. Cancers can “shape-shift” to evade drug treatment. As explained above, one way they do this is by jumping binary classes. The goal, therefore, is to have efficient treatments that target both classes. Blocking both types of cancers would prevent class switching, possibly meaning that cancer cells would have nowhere to hide. 

KR: Are there any limitations to this work that you wish to highlight? What additional research are you planning to conduct in this area?

In vivo drug and synergy studies. Our drug data is limited to in vitro studies, thus it will be essential to test those drugs with in vivo preclinical animal models. It is also limited to single treatments, and so it will be critical to search for drug synergies. As we identified multiple selective drugs, the first approach will be to try combinations of those agents. Large scale screens may identify more potent synergistic drug combinations.

How is the YAP/PC1+ program regulated? We showed that silencing of YAP and the PC1+ genes is epigenetic. A key issue for the future is to deduce the underlying mechanism of epigenetic silencing. That knowledge should throw light on how to reactivate the YAP-dependent anti-cancer program in YAPoff tumors. Several YAPoff cancers are extremely lethal (e.g., SCLC, or end-stage drug resistant neuroendocrine prostate cancer) and there are no good therapies, so there is considerable interest in devising new approaches. Understanding the regulation of these genes is also critical to deduce how cancers switch from one class to the other, and to devise ways to prevent that jump.

Does YAP prevent the YAPon to YAPoff class switch? While we showed that YAPon cells can switch to the YAPoff state to resist therapy, we did not ask whether YAP down-regulation is required for this jump. We presume that it is, because, as explained above, the end-state YAPoff cancer hates YAP. We need to exploit systems that will allow us to catch the class-conversion in the act to directly test if YAP down regulation is critical.

Do cancers switch binary classes in both directions? We showed that YAPon cancers can switch to the YAPoff type, but this raises the intriguing issue as to whether the reverse is true. Can YAPoff cancers evade therapy by jumping to a YAPon state? There is already some evidence in the literature that this is indeed the case, so we’re pursuing this question vigorously.

Does YAP inhibit blood cancers, and if so, how? All the functional studies in our paper focused on solid YAPoff cancers, which are neural and neuroendocrine in nature. However, blood (“liquid”) cancers are also YAPoff, and our in vitro analyses show they share drug and genetic sensitivities, and of course, they also grow in suspension. Thus, a major area is to define whether and how YAP affects these cancers, and whether this insight can be used to improve treatments for leukemia and lymphoma.

Rod Bremner was speaking to Kate Robinson, Editorial Assistant for Technology Networks