At the beginning of drug treatment, tumor cells have two ways — to remain sensitive to therapy (and die) or to become resistant (resistant).
It is believed that the drug resistance of tumor cells arises due to new mutations. For example, mutations that change the receptor protein on the cell surface in one way or another can make it "invisible", since the drug will no longer be able to interact with its target. In other cases, tumor cells, as a result of mutations, find a workaround for important signaling pathways that were turned off by previous therapy. There may be different options. Usually, in such cases, the patient's treatment regimen is changed — other drugs are used that will be effective, taking into account the new mutation. However, there is another strategy that can be used to counteract the effects of drugs, and this strategy is not related to mutations, but to the amazing ability of tumor cells to adapt to changing environmental conditions. At the same time, there are no changes in DNA, only the activity of genes (epigenetic changes) changes — some begin to work harder, some weaker. Surprisingly, this leads to the appearance of drug—resistant clones (daughter tumor cells that survived treatment), which in the future, even after successful treatment at first glance, will ensure the occurrence of relapses and metastasis of the tumor. As a result, antitumor therapy begins to resemble an exhausting battle with the Lernaean hydra — while you chop off one head, another grows and you have to dodge it. To win in a fight with such a serious opponent, it is necessary to thoroughly study the mechanisms of resistance to antitumor effects. This is exactly what many research groups around the world are doing today.
Unfortunately, sooner or later most tumors become resistant to the drugs used: relapse occurs, metastases appear, in other words, the disease progresses.
How does resistance arise?
At the first contact of the tumor with the drug, active cell death begins. With chemotherapeutic effects, actively dividing cells die first of all. If targeted drugs are used, those cells that carry the genetic disorder corresponding to this drug die. Obviously, the more tumor cells die, the better the clinical effect of the antitumor drug will be. The resumption of tumor growth against the background of ongoing treatment or the appearance of new foci in other organs is usually interpreted in clinical practice as a manifestation of resistance.
There is a distinction between primary and secondary (acquired) resistance.
Primary resistance occurs in cases where some characteristics inherent in tumor cells initially ensure its resistance to any drug. Thus, some rare EGFR mutations are associated with primary resistance to tyrosine kinase inhibitors in non-small cell lung cancer.
Secondary or acquired resistance occurs when initially drug-sensitive tumor cells become resistant to therapy already during treatment. Despite the differences between primary and acquired resistance, their biological mechanisms are largely similar and are due to the clonal diversity existing in the tumor. All tumors, in addition to a few driver mutations, carry many other passenger mutations. In any case, some of the tumor cells experience lethal effects on all other cells. Only with primary resistance do such resistant cells exist in the tumor initially, and with acquired ones they arise by selection from the entire spectrum of mutations in the selection process of those that provide an undeniable competitive advantage in the current conditions. For example, the initial presence in a small percentage of the tumor population of a mutation in exon 20 of the EGFR gene, designated as T790M (which means the replacement of threonine by methionine (M) at position 790 of the protein), probably does not give any advantage relative to other cells in the absence of therapy, when exposed to the ITK EGFR 1st and 2nd generations (gefitinib, erlotinib, afatinib) provides cells with a selective advantage simply due to the fact that there is no target for the antitumor drug in this case . The selection of such a pre-existing clone leads to the rapid replacement of all other tumor clones with a drug-resistant clone of cells with the T790M mutation.
Thus, under the condition of clonal diversity of the tumor, targeted therapy acts as a guiding factor in evolution, leading to the emergence of resistance. Clinically, such tumors are characterized by a short-term response to treatment, followed by a steady growth and progression of the disease. There is a third—generation drug, osimertinib, which is used in the treatment of patients with NSCLC with the T790M mutation, but resistance to it sooner or later arises. It can be assumed that other mutations appear in the tumor, which lead to the activation of alternative signaling pathways (for example, amplification of Her2/neu and MET). However, recently there has been increasing evidence that resistance mechanisms can be not only genetic, but also non—genetic - that is, cells manage to survive without mutations, without changes in the genetic text.
Scientists have known before: what doesn't kill cancer makes it stronger. And this is not always due to mutations. There are well-known examples of changes in the phenotype of tumor cells as a mechanism for the development of drug resistance. Thus, the phenomenon of transformation of non-small cell into small cell cancer during treatment with osimertinib is described. This is a prognostically unfavorable form of the disease, for which there are also no effective treatment regimens. Similarly, prostate cancer (prostate cancer) cells can undergo transdifferentiation (transformation of cells of one type into another) into an extremely aggressive neuroendocrine phenotype when androgen deprivation (aimed at reducing androgen levels) therapy is prescribed. Prostate cancer cells acquire the phenotypic features of neurons, their shape becomes more elongated, secretory granules appear in the cytoplasm, which is accompanied by an increase in the expression of neuroendocrine markers chromogranin A and synaptophysin. At the same time, the number of neuroendocrine cells increases as the duration of treatment increases. A feature of the neuroendocrine phenotype is that such cells do not contain androgen receptors. As in the case of non-small cell lung cancer, the phenotypic transformation of prostate cancer cells does not affect the genotype, but is an adaptive mechanism that allows tumor cells to remain viable under adverse conditions of therapeutic exposure.
There is another phenomenon that forces scientists to consider phenotypic changes as a serious player in the development of tumor resistance. We are talking about the reversibility of resistance, which has been repeatedly described in various clinical studies. Thus, it was observed that in some cases of NSCLC, a break in treatment "returned" the tumor cells that had become resistant to the action of TIC back to their original drug-sensitive state. The reversibility of drug resistance has also been described in a human melanoma model transplanted into mice. It was shown on such xenografts that "therapeutic vacations" during exposure to vemurafenib prevented the development of resistance in immunocompetent animals. These data suggest that acquired drug resistance may include a reversible phase. Since genetic changes are usually not reversible, the phenotype is again under suspicion. This leads to the conclusion that the mechanism of resistance to therapy is not always associated with changes in the DNA sequence, but may also be determined by more plastic patterns of activity (or, as biologists say, expression) of individual genes.
The emergence of new phenotypic cell conditions helps the tumor to withstand suddenly changing environmental conditions and, along with genetic heterogeneity, plays an important role in tumor progression and the formation of resistance to various antitumor therapeutic effects. Each tumor cell in the body is unique in its potential to undergo various changes, but even one of the many that has escaped the effects of therapy by changing the phenotype can potentially cause disease progression and become the basis for the formation of drug resistance.
The criminal is armed and very dangerous, or What is hidden behind the abbreviation DTP
DTP (Drug-Tolerant Persist) is a persistent cell condition that is induced by exposure to a drug under the condition of tolerance to it. Due to the difficulty of translation, hereafter we will refer to this state of cells by its English abbreviation. Note also that tolerance should be distinguished from resistance. Resistance refers to the ability to resist the effects of a particular drug. The development of resistance is a multi—stage process and one of its "steps" is tolerance. Tolerance is a decrease in the reaction to repeated administration of a particular drug, which requires an increasing and increasing dose to achieve an effect. Tolerant cells enter a state with reduced or shut down metabolism and thus become persistent.
The term DTP was first used by Professor Srinath Sharma and co-authors in 2010, when he described the transformation of a small portion of tumor cells into a condition in which previously sensitive to the drug erlotinib cell lines of non-small cell lung cancer became resistant to it. At a dose of erlotinib corresponding to that used in standard therapy regimens, after 9 days of continuous exposure to the drug, 0.03% of cells remained in the studied cell lines, which were predominantly in the G1 phase of the cell cycle and were characterized as drug-tolerant persisters (DTP). After some time, about 20% of them passed into a slowly proliferating state and formed a growing population of drug-tolerant expanded population (DTEP) cells. Both phenotypic variants had pronounced resistance to ITK — even a very high concentration of the drug did not lead to apoptosis (cell death). Interestingly, no T790M mutation or MET amplification was detected in these cells (doubling of a DNA region of a gene, leading to an abnormally stronger stimulation of the gene and tumor growth) as standard mechanisms of acquired resistance. The resulting cells were insensitive to many classes of targeted drugs, but, in addition, they were significantly less resistant to the chemotherapeutic drug cisplatin, which suggests a non-specific nature of resistance.
Since the release of this key publication, the data of Sharma and co-authors have been repeatedly confirmed by many other researchers. The existence of a population of DTP cells has been confirmed in glioblastoma (a malignant brain tumor), melanoma (a malignant tumor arising from melanocytes), colorectal cancer, breast cancer, and many other tumors.
As can be seen from Figure 2, the DTP population is very small. But it is the presence of these cells that protects the tumor from complete death when exposed to drugs. By keeping DTP in reserve, the tumor has every opportunity to launch a new attack on the body, and it is only a matter of time before this happens.
The first is an introduction to resistance
Interestingly, the process of formation of a small number of cell generations capable of maintaining viability in the presence of drugs (in concentrations many times higher than therapeutic ones) was described back in the 40s of the twentieth century. However, it was not molecular biologists who did this, but microbiologists who noticed that there was no complete sterilization effect in the culture of bacterial cells of Staphylococcus aureus when exposed to penicillin. A small surviving subpopulation of bacteria remained viable, and after the cessation of antibiotic exposure, it gave rise to a new microbial population — by the way, also sensitive to it. This observation became convincing evidence that the new bacterial population retains the biological properties of the maternal culture and does not represent a genetically modified penicillin-resistant strain, and the surviving cells are a phenotypic variant of the original population. Bacterial cells that remain viable in the presence of an antibiotic are called persistent. They are in a state of metabolic and reproductive rest, which was confirmed two years later, in 1944, in the experiments of Joseph Bigger. Since the discovery, the phenomenon of bacterial persistence has been actively studied. In particular, it was found that persister cells usually make up only a small part of the bacterial population. In wild—type Escherichia coli strains, their frequency is no more than one per million cells. But in some conditions, for example, in biofilms, the frequency of such persisters can increase significantly, more than a hundred times. The biological feasibility of the existence of such cellular generations of microorganisms is associated with the need to preserve the population in the face of changing environmental conditions. Characterized by reduced metabolic activity and being in the resting phase, persister cells acquire the ability to quickly restore their initial pathogenic characteristics under optimal conditions. From these positions, a population of bacterial cells with persisters can be considered as having a certain adaptive potential aimed at preserving the species. Since the first publication on DTP appeared, interest in the phenomenon of tumor cells that remain viable during treatment and are able to change their phenotype in the presence of a stressful factor in the environment has increased many times. Persistent tumor cells are being studied by scientific laboratories all over the world. By searching for drug-tolerant persistent cells, you can find almost 1,500 different works in the PubMed database. Sessions at various conferences are devoted to the DTP phenomenon. Unfortunately, DTP cells are able to skillfully disguise themselves. On the one hand, the population of persisters is enriched with cells synthesizing stem cell markers — CD133 and CD44. Cells with this immunophenotype are characterized by a gene expression profile that is largely similar to that of stem cells in normal tissues. On the other hand, cells in the DTP state acquire the features of aging cells, that is, cells that have ceased to perform their functions, but have not yet died. Perhaps the tumor cells are only "pretending" to age in order to wait out the stress caused by drug therapy. And then they "get younger" again and begin to multiply, which inevitably leads to resistance and tumor progression. However, such reversible aging is a rare phenomenon. In addition to the fact that senescent cells perform their functions worse, they also acquire a special secretory phenotype SASP (from the English Senescence associated secretory phenotype) and secrete many soluble factors into the extracellular environment, such as cytokines, chemokines, proteases and growth factors. On the one hand, the profile of SASP products secreted by senescent cells attracts cells of the immune system, which underlies tumor suppression. On the other hand, SASP components stimulate the proliferation of cells in the microenvironment, including nearby DTP. Which will ultimately outweigh the positive or negative effects of SASP will depend on the balance between the time senescent cells are present in the population and the rate of their elimination by cells of the immune system. In this regard, it may be promising to develop strategies aimed at modulating the composition of factors secreted by senescent cells. Thus, the DTP population, although small, is heterogeneous in its composition and may vary depending on the type of tumor, as well as vary at different stages of its development and depending on the treatment regimen used. These malignant cells demonstrate high plasticity potential, resistance to stress factors, for example, to the action of anticancer drugs, and are characterized by a reversible resting state as a general response to stress. At a certain point in time, new mutations appear in the descendant cells of those that have experienced drug exposure and have entered a state of tolerance to it, and in an amount significantly greater than in the "parent" cells. This has been demonstrated in experiments on DiFi and WiDr colorectal cancer cell lines. The possible mechanism of occurrence of such mutations is most likely related to errors in the replication process, since against the background of suppression of repair genes in DTP cells, error-prone DNA polymerases are activated, which leads to inappropriate mutagenesis. Ultimately, it is thanks to DTP that the tumor is able to survive drug exposure, gradually resume growth, acquire new genetic aberrations and become resistant, and this resistance will be irreversible. Now, after the discovery of DTP, the progression of malignant neoplasms seems to be an even more complex process, and antitumor therapy is even more problematic. Nevertheless, there are strategies for dealing with DTP cells. For example, you can try to "wake them up" by increasing the number of proliferating cells, and then use, for example, cytostatic therapy. The disadvantage here is that by "letting the genie out of the bottle," we can not effectively control it, which is fraught with rapid recurrence or metastasis of the tumor. Another approach is to keep DTP cells in a state of constant hibernation and prevent them from "waking up." However, this requires constant exposure of therapeutic drugs to the tumor and the body as a whole, which is impossible in real life, primarily due to the risk of adverse events. In any case, we understand that there are no simple ways to treat malignant diseases — cancer cells have too many tricks to help them survive. Building an optimal treatment regimen in order to prevent the formation of a resistant tumor clone requires the development of complex combination therapy, which should also be aimed at cells in the DTP state. Overcoming tumor resistance is a serious challenge facing modern medical science. The discovery of DTP cells, the identification of their functioning mechanisms, as well as the first positive results in the development of strategies to combat them allow us to look to the future with some degree of optimism.