This article is reprinted with permission - Source: Back to the Park (ID: fanpu2019); Written by | Samuel F. Bakhoum (Memorial Sloan Kettering Cancer Center); Compiled by | Kestrel; Back to the Park (ID: fanpu2019) Public.
There are still too many mysteries in the fight between cancer cells and the immune system. Each quest brings new hope.
Seven years have passed since then, but the feeling of loss is still clear.
It was a day in 2015 when I got the DNA sequencing results of a patient's tumor tissue; her lung cancer had metastasized to the brain, but I couldn't find half of the genetic alterations in the report that would indicate a therapeutic target, and at the same time, something else caught my attention: the data showed that almost every chromosome had undergone many changes in structure and number.
In normal cells, each chromosome has two copies, but not in cancer cells, where the number of copies varies from one to five or six, and sometimes chromosome fragments are present. Although cancer researchers and clinicians have known for a long time that CIN is a sign of advanced cancer or metastasis, I have never seen a patient like this one, who is only 59 years old, with such a serious condition. I have never seen such a serious case like this patient, who is only 59 years old, not to mention that she has only been diagnosed recently, while CIN is generally considered to occur gradually over a relatively long time span.
Oncologists are at their wits' end because there are no good targeted therapies for CIN and patients are simply subjected to multiple rounds of systemic chemotherapy and radiation. My patient was no different, and in addition to the usual side effects, she had to suffer from neurotoxicity from the radiation to the brain, which could cause memory loss and other cognitive deficits.
Such a dilemma reminds me of my own background in cell biology. In the past, my scientific work during my PhD was to figure out how chromosomes are equally distributed during mitosis in normal cells. This process is intricate, but it plays out every day in many tissues. Living organisms have evolved multiple backup mechanisms to ensure that this process does not go wrong. In the unlikely event that something goes wrong, cells with abnormal chromosome numbers are quickly removed. This is not the case with cancer cells, which are highly tolerant to chromosomal abnormalities and where genetic changes on such a large scale are closely associated with disease progression. However, it is not clear whether and how CIN plays an active role in tumor progression and metastasis.
It wasn't until a few years ago that I started working on whether CIN drives cancer development or is just a phenomenon that accompanies it. To do this, I started a collaboration with Lewis Cantley, then director of the Meyer Cancer Center at Weill Cornell Medicine and now at the Dana Farber Cancer Institute. We genetically manipulated a variety of chromosomally unstable and metastatic cancer cells to reduce their CIN levels without affecting the other genetic abnormalities they carry. It became clear that the cancer cells that lost their CIN also lost their ability to metastasize. One other thing that surprised us was that we found that CIN promotes cancer metastasis by causing chronic inflammation. So, ultimately, it is the body's own immune response that allows cancer cells to break away from the primary tumor and invade other organs.
In 2018, our work was published in the journal Nature (). The findings suggest to us that the step of making the genetic material unstable is itself crucial for the evolution (evolve) of the cancer. This could be a starting point for new therapeutic ideas: can we find targets in this step to treat cancers with severe CIN conditions? Can we find ways to stabilize the genome or reduce the chronic inflammation caused by CIN to stop cancer metastasis? Can we modify the immune system to remove cells with abnormal chromosome numbers?
To address these key questions, my laboratory at Memorial Sloan Kettering Cancer Center (MSK or MSKCC) employs an interdisciplinary, cell biology-based approach that combines single-cell genomics, mathematical modeling, and clinical sampling. We believe that by integrating these approaches, we will understand how CIN modifies the behavior of cancer cells and enhances their fitness to sustain cancer progression. Further, we aim to uncover which cellular pathways allow cancer cells to tolerate CIN and then treat cancer by targeting those pathways.
Also in 2018, I co-founded Volastra Therapeutics with Cantley and another colleague, Olivier Elemento, with a view to complementing each other's academic work on CIN. The company's scientific staff is developing therapies targeting CIN for a variety of cancers. Through such extensive collaborations, we hope to explore and develop new therapeutic approaches for patients with chromosomally unstable cancers.
Overlooked signs of cancer
Since scientists measured the first cancer cell genome in 2006, we have been gaining insight into what genetic material is altered to drive cancer development and progression. Scientists have developed targeted therapies that act on the genes that promote tumor development. The concept of these therapies is based on the assumption that if we can suppress these genes, the tumor will stop progressing. Therefore, we have to sequence the genome of tumor tissue to find out the cancer-promoting genes in each patient, a step that has become a routine practice for many oncologists in MSK. However, the limitations of individualized tumor therapy become apparent when sequencing fails to help us find the target: there are indeed some successful cases, but the results are still very limited for most patients with advanced cancer.
Even if targeted therapies can be useful, they may only be effective at first, as tumors "evolve". They can often escape our drugs. One of the most powerful weapons of cancer cells is CIN, and every time they divide - thanks to CIN - their chromosomes undergo random rearrangements. As a result, errors in chromosome segregation accumulate, leading to a high degree of heterogeneity in chromosome composition and copy number among the cells in the cancerous tissue. This phenomenon is called "aneuploidy". Indeed, advanced tumors that recur after multiple rounds of treatment are characterized by a high degree of chromosomal instability and aneuploidy, and in such tumors, drugs that suppress a single mutated gene are no longer effective, despite the fact that they have led to cancer improvement.
Researchers have known for decades that aneuploidy is a feature of human cancer, but it was not until 1997 that Christoph Lengauer and Bert Volgelstein of Johns Hopkins University School of Medicine (JHUSM) first demonstrated the role of CIN in promoting cancer cell heterogeneity. Through their work, it was soon understood that CIN has the potential to stimulate tumor evolution and progression: CIN is able to regulate chromosome copy number and therefore the copy number of genes on these chromosomes. Recently, Stephen Elledge of Harvard Medical School found that human malignancies do indeed increase the copy number of chromosomes with oncogenes and decrease the copy number of chromosomes with oncogenes, thereby increasing their own fitness.
Despite the importance of CIN for human cancer, laboratories focus on genetic mutations. The methodological revolution brought about by next-generation sequencing technologies has led the academic community to focus all attention on the contribution of individual genes to carcinogenesis and has led to many important discoveries that have expanded our understanding of the role played by many genes in tumorigenesis. However, this approach also has drawbacks in that it ignores large scale chromosomal aberrations and their effects on gene function and cancer cell behavior. Purifying DNA from whole tumor tissue samples and sequencing it certainly gives us a clearer view of the genetic information on chromosomes, but it does not allow the localization of DNA fragments with altered sequences to chromosomes and obscures the heterogeneity of chromosome copy numbers between cells.
In the past decade, researchers have begun to focus more attention on large-scale chromosomal alterations, and in 2010, Robert Benezra of MSK and others published a major study showing that tumors that have acquired CIN are no longer dependent on the oncogene that triggered the cancer in the first place. Specifically, when researchers upregulated the oncogene KRAS to induce lung cancer in mice and then removed the upregulation, tumor regression was observed; however, if genetic engineering was used to further artificially introduce CIN into the cancerous cells, tumor regression was not observed. So-called targeted therapies are designed to specifically inhibit oncogenes like KRAS to prevent them from driving tumorigenesis, but malignant tumors gradually become resistant to such therapies, and this work tells us how resistance arises.
Since then, Charles Swanton's group at University College London and the Crick Institute has given strong evidence that CIN is important in human cancers. in 2017, by following lung cancer patients, Swanton's team demonstrated that it is chromosomal instability - rather than the number of point mutations in the tumor genome - was associated with reduced overall survival. Their subsequent studies showed that CIN likely plays an important role in all aspects of tumor biology, whether it is tumor metastasis or cancer cells escaping immune surveillance, and that the progressive change in chromosome copy number that accompanies each division of cancer cells provides tumors with the ability to evolve under a variety of selective pressures.
Because these scientists continue to study the role of CIN in cancer, the boundaries between cancer genomics and cell biology are gradually dissolving. On the one hand, the genetic code carried by chromosomes can be deciphered by sophisticated genomic means; on the other hand, chromosome life cycles and segregation behavior during cell division can essentially be tracked with high-resolution light microscopy. For example, chromosomes that make mistakes during segregation end up in a "micro-nuclei" that hold small segments of DNA and are separated from the nucleus - the "master nucleus The micro-nuclei are separated from the nucleus - the "main nucleus". The micro-nuclei have long been seen as a feature that distinguishes cancer cells from surrounding normal tissue. The work of several groups has shown that the nuclear membrane that encases the micronuclei often breaks down, spilling chromosomes into the cytoplasm, where they are exposed to nucleases and degraded, like other proteins, and become fragmented.
Figure 1. Micrographs of micronuclei. The small black dots in the cell are the micronuclei. (https://doi.org/10.1016/B978-0-12-800764-8.00006-9)
After a massive chromosome break occurs, some fragments are lost and others are randomly joined together, and then the direction and order in which the fragments are joined to each other can be wrong, resulting in the formation of new, highly abnormal chromosomes. This process is called chromothripsis. Recently, researchers have found that chromothripsis is an important mechanism that stimulates cancer progression. In addition to stepwise changes in chromosome number, through massive chromosomal rearrangements, the fragmented chromosomes soon form a pool of prizes that can open up cancer - which is already serious when it occurs. This process has the potential to rapidly amplify oncogenes and lose oncogenes. We also now know that chromosome fragmentation may place oncogenes near highly active chromosomal regions and also promote the formation of circular extrachromosomal DNA, both of which contribute to the rapid development of tumor cells that are resistant to targeted therapies.
Although researchers identified the disorganized state of chromosomes in cancer early on, it was not until the advent of state-of-the-art genomics and microscopy that our understanding of the chromosomal fragmentation process became clear. in 2015, David Pellman and colleagues at the Dana-Farber Cancer Institute applied microscopy to capture individual cancer cells that showed chromosomal segregation errors and performed genomic analysis. With this approach (which they called Look-seq), the researchers showed how complex patterns of chromosomal rearrangements, common in the human cancer genome, emerge within a single cell cycle. Through a variety of pathways, CIN appears to promote both progressive and intermittent bursts of tumor genome evolution. This, presumably, is what happened in my patient - a massive chromosomal aberration occurred not long after diagnosis.
Mechanism of micronucleus formation
During cell division, there are many chromosome segregation errors that can lead to micronucleus formation. Even if the chromosomes do not eventually segregate incorrectly, micronuclei may still form. For example, in the case shown in Figure 2, although the chromosomes are eventually divided equally between the two daughter cells, the left daughter cell forms a micronucleus due to a lag in segregation. These events are not mutually exclusive or independent, but simply exacerbate chromosome disorganization each time they occur.
Figure 2. Mis-attachment.
When microtubules from both poles of the dividing cell attach to the same centriole (top), the segregation of the attached chromosome lags behind the other chromosomes and is often later wrapped into the micronucleus, even though it eventually divides to the cell where it should go (bottom left).
Figure 2. Non-integer multiplicity.
If chromosome segregation does go wrong, whether due to microtubule misattachment or for some other reason, the missegregated chromosome may be wrapped in the nuclear membrane with the other chromosomes. If it is not wrapped, the aneuploid cell that forms may carry a lagging chromosome, increasing the risk of micronucleus formation.
Figure 3. Chromosome fusion.
Telomere shortening or damage can make chromosome fusion more likely to occur. Such a fusion event produces chromosomes with two centromeres. During the next cell division, the chromosomes with the double centromere are torn apart and split into two daughter cells. These damaged chromosomes are either immediately isolated to the micronucleus or are still isolated to the micronucleus during the next cell division because they cannot complete replication properly.
CIN and inflammation
In studying how chromosomal instability affects cancer metastasis, we made an unexpected discovery. Specifically, cancer cells that have developed CIN have an activated inflammation-related signaling pathway, which allows the cancer cells to produce and secrete a variety of inflammatory factors associated with cancer metastasis. This was a mystery at first, because these cells were only kept in the laboratory and were not grown in experimental animals, so they did not have any exposure to immune cells. So what triggers these "inflammatory responses"?
After a long search in the microscope, we not only observed that micronuclei dominate the cells that develop CIN, but also that those cells that contain ruptured micronuclei carry an immune-related enzyme called cGAS. cGAS was first discovered in 2013 by James Chen of the University of Texas Southwestern Medical Center. Chen, a double-stranded DNA receptor localized in the cytoplasm, was first discovered in 2013. We then envisioned that micronucleus rupture and the ensuing exposure of chromosomes in the cytoplasm might be recognized by cancer cells as a red flag, just as cells recognize invading pathogenic DNA. Of course, we then confirmed that the ruptured micronucleus was capable of powerfully activating cGAS and its associated protein STING, which in turn activates the intrinsic immune response. But unlike acute viral infections that clear within a few days, micronuclei rupture event after event in the cytoplasm of cancer cells, resulting in a constant activation of inflammatory pathways and ongoing inflammation.
At this point, things are clear: the cancer cells must have utilized some protective immune pathway to be able to break through these inflammatory defenses. Although activation of intrinsic immune signaling pathways may protect the body from tumor emergence in the early stages of tumorigenesis, at a certain stage, tumor cells are able to cross over these protective mechanisms, tolerate the inflammatory response triggered by CIN, and gradually use these pathways to drive tumor growth. The ability of cancer cells to maintain inflammation is critical to their ability to metastasize to another organ. Immune cells are the most mobile cells in the body, and within hours after the body encounters an infection or develops a wound, they are able to migrate across the vascular system to inflamed tissues with elevated hydrostatic pressure and reach the damaged area. It is by mimicking this physiological process that cancer cells use CIN and other genomic abnormalities to achieve metastasis.
The association between chronic inflammation and cancer has a long history. In fact, the most central features of inflammation described by the ancient Roman encyclopedist Aulus Cornelis Celsus apply to cancer - redness, heat, painful swelling, and for centuries, clinicians have often referred to tumors as "unhealable wounds "because it is constantly inflamed. It is not entirely clear what role inflammatory signaling pathways play in the development of cancer, but correlating intrinsic genomic abnormalities (e.g., chromosomal instability) with persistent inflammation in cancer reveals that CIN not only drives genetic heterogeneity in tumors, but also stimulates cancer metastasis through non-genetic mechanisms (i.e., mimicking the inflammatory response).
How does micronucleus rupture promote cancer progression?
Figure 4. Micronucleus rupture promotes cancer development.
The nuclear membrane of the micronucleus is brittle and often ruptures, causing chromosomes to scatter into the cytoplasm. The chromosomes inside the cytoplasm are cut into small fragments by nucleases, which are either lost, randomly joined together, or linked end-to-end to form a loop of extrachromosomal DNA, a process known as "chromosome fragmentation. The complex rearrangement of chromosomes that results from this process drives tumor production.
Meanwhile, DNA lodged in the cytoplasm triggers the cGAS-STING inflammatory pathway. It is believed that this pathway evolved from the mechanism of resistance to viral infection. cGAS binds to DNA dispersed by micronucleus rupture and catalyzes the production of 2'3'-cyclic guanosine adenosine monophosphate (cGAMP), which activates STING proteins and downstream inflammatory pathways. Since there are many micronuclei in cancer tissues, it is possible that this pathway is consistently activated, triggering a sustained inflammatory response that drives tumor growth and metastasis.
Targeting CIN
Unlike cancer cells, normal cells cannot tolerate chromosome segregation errors. Research led by the late Angelika Amon of the Massachusetts Institute of Technology (MIT) had found that aneuploidy is associated with multiple cellular defects, such as metabolic disorders and mitochondrial dysfunction, as well as cellular stress responses induced by protein folding errors. Indeed, the body has evolved multiple mechanisms that can eventually clear aneuploid cells. Duan Compton and others at the Geisel School of Medicine at Dartmouth found that in response to chromosome segregation errors, normal cells rapidly activate the tumor suppressor p53, which stops cell division and prevents the spread of aneuploid cells. These important protective mechanisms are designed to maintain the integrity of the genome, and generally with them, the genome is fine. But in cancer cells, these lines of defense are breached. Therefore, understanding how tumor cells respond to CIN may shed light on the treatment of cancer.
The academic community has shown increasing interest in the mechanisms by which tumor cells tolerate CIN. Recently, several groups have identified, through genetic screens, a number of genes and some cellular activities that are indispensable for tumor cells with high levels of CIN and whose absence is lethal. One of them is the kinesin Kif18a, which plays a role during mitosis, during chromosome movement. For cancer cells with CIN, the Kif18a protein is essential for cell division, but not for cancer cells without CIN. Interestingly, if a mouse lacks a functional Kif18a protein, it can still survive, but exhibits only minor defects. This kinesin could then potentially be a safe and effective therapeutic target. There is now a phase I clinical trial testing the efficacy of Kif18a inhibitors in patients with advanced cancer.
Several other groups are exploring alternative therapeutic strategies, namely, inhibition of targets that allow tumor cells to overcome chronic inflammation. For example, the ENPP1 protein, which was initially identified by Timothy Mitchison at Harvard Medical School and Lingyin Li at Stanford University (also at Harvard at the time), was later found by our group to be selectively upregulated in chromosomally unstable cancer cells. The degradation of extracellular cGAMP prevents immune cells from detecting cancer cells. cGAMP degradation also produces adenosine, which in turn exacerbates immune disorders and promotes cancer cell migration. We are impressed by the ability of cancer cells to turn enemies into friends and to appropriate the protective mechanisms of the immune system for their own use.
Volastra researchers are continuing to develop a deeper understanding of the biological mechanisms of CIN, which, combined with computational and genetic screening approaches, is slowly leading to the discovery of a number of cancer treatment strategies. The current top drug candidate targets the process of microtubule binding to chromosomes, which selectively kills chromosomally unstable cancer cells without injuring other cells. The drug is scheduled to be advanced to clinical trials in 2023. Other therapeutic strategies we are exploring include regulating spindle formation, altering the organization of chromosomes during cytokinesis, and harnessing the CIN-driven inflammatory response to fight cancer.
CIN is an attractive drug target because only cancer cells exhibit chromosomal instability, and then treatment for CIN can do no wrong - the holy grail of cancer therapy. The Holy Grail of cancer therapy. Over the past decade or so, the fields of cell biology, genomics, and cancer biology have become progressively intertwined, with interdisciplinary research approaches and collaborations between academia and industry leading to new discoveries. The ultimate goal of all this is to serve patients - like my patient whose tumor has metastasized to the brain, and others for whom the available treatment options are very limited.
This article was compiled with permission from the-scientist.com and was originally published under the title
Read the original article at
https://www.the-scientist.com/features/how-chaos-in-chromosomes-helps-drive-cancer-spread-69695
