Cancer cells can dodge chemotherapy by entering a state of senescence


Cancer cells can dodge chemotherapy by entering a state that bears similarity to certain kinds of senescence, a type of “active hibernation” that enables them to weather the stress induced by aggressive treatments aimed at destroying them, according to a new study by scientists at Weill Cornell Medicine.

These findings have implications for developing new drug combinations that could block senescence and make chemotherapy more effective.

In a study published Jan. 26 in Cancer Discovery, a journal of the American Association for Cancer Research, the investigators reported that this biologic process could help explain why cancers so often recur after treatment. The research was done in both organoids and mouse models made from patients’ samples of acute myeloid leukemia (AML) tumors.

The findings were also verified by looking at samples from AML patients that were collected throughout the course of treatment and relapse.

“Acute myeloid leukemia can be put into remission with chemotherapy, but it almost always comes back, and when it does it’s incurable,” said senior author Dr. Ari M. Melnick, the Gebroe Family Professor of Hematology and Medical Oncology and a member of the Sandra and Edward Meyer Cancer Center at Weill Cornell Medicine. “

A longstanding question in the field has been, ‘Why can’t you get rid of all the cancer cells?’ A similar question can be posed for many other types of aggressive cancer in addition to AML.”

For years, cancer researchers have studied how tumors are able to rebound after they appear to be completely wiped out by chemotherapy. One theory has been that because not all cells within a tumor are the same at the genetic level – a condition called tumor heterogeneity – a small subset of cells are able to resist treatment and begin growing again.

Another theory involves the idea of tumor stem cells – that some of the cells within a tumor have special properties that allow them to re-form a tumor after chemotherapy has been given.

The idea that senescence is involved does not replace these other theories. In fact, it could provide new insight into explaining these other processes, Dr. Melnick said.

In the study, the researchers found that when AML cells were exposed to chemotherapy, a subset of the cells went into a state of hibernation, or senescence, while at the same time assuming a condition that looked very much like inflammation. They looked similar to cells that have undergone an injury and need to promote wound healing – shutting down the majority of their functions while recruiting immune cells to nurse them back to health.

“These characteristics are also commonly seen in developing embryos that temporarily shut down their growth due to lack of nutrition, a state called embryonic diapause,” Dr. Melnick explained. “It’s not a special process, but normal biological activity that’s playing out in the context of tumors.”

Further research revealed that this inflammatory senescent state was induced by a protein called ATR, suggesting that blocking ATR could be a way to prevent cancer cells from adopting this condition.

The investigators tested this hypothesis in the lab and confirmed that giving leukemia cells an ATR inhibitor before chemotherapy prevented them from entering senescence, thereby allowing chemotherapy to kill all of the cells.

Importantly, studies published at the same time from two other groups reported that the role of senescence is important not just for AML, but for recurrent cases of breast cancer, prostate cancer and gastrointestinal cancers as well. Dr. Melnick was a contributor to one of those other studies.

Dr. Melnick and his colleagues are now working with companies that make ATR inhibitors to find a way to translate these findings to the clinic. However, much more research is needed, because many questions remain about when and how ATR inhibitors would need to be given.

“Timing will be very critical,” he said. “We still have a lot to work out in the laboratory before we can study this in patients.”

The rapid development of novel therapeutic strategies, represented by targeted therapy, has made great contributions to the improvement of clinical outcomes in patients with cancer.1,2 However, such improvements have not been translated into complete remission (CR) due to the inevitable emergence of drug resistance, which is regarded as a major impediment in clinics for achieving complete cures.1,3

For decades, along with the identification of various resistance-conferring mutations, researchers have theorized that this therapeutic failure is mainly attributable to genomic mechanisms, such as the acquisition of mutations that occur on the drug target, thus impairing the drug binding and mutation-induced continuous activation of pro-survival pathways.4,5

This would suggest that reagents designed to selectively repress such bona fide resistance mechanisms hold great promise for the realization of long-term curative effects and the improvement of living quality in patients with cancer. However, drug resistance frequently occurs and remains a clinical challenge.6,7

The development of secondary mutations may also provide a mechanistic explanation for such resistance, and may even present a treatment option for patients (e.g., the so-called “next-generation” tyrosine kinase inhibitor [TKI] for non-small cell lung cancer [NSCLC] patients).8

The observation that clones with resistance-conferring mutations can pre-exist within an individual tumor prior to drug exposure and be further selected during treatment indicates that merely targeting the validated genetic resistance mechanisms is not enough.9–12

Occurring in parallel are numerable cases that are not related to genomic/genetic alterations, raising the possibility of non-mutational mechanisms involved in maintaining cancer cell survival and growth upon treatment.13–16 For instance, a rare subpopulation of cancer stem cells (CSCs), or poorly differentiated cancer cells equipped with enhanced drug efflux properties and heightened self-renewal potential, is intrinsically more refractory to multiple cancer therapies, suggesting a fundamental role of CSCs as a reservoir for tumor recurrence.17

Indeed, such stem cell-like phenotype-dependent relapses have been previously described in patients with chronic myelogenous leukemia following imatinib mesylate treatment18,19 and have been further documented in various types of solid tumors.20–22 Being regarded as the source of non-mutational resistance, this subpopulation—named drug-tolerant persisters (DTPs), has been widely recognized for its dormant, slow-cycling state and stem-like signature.13

Such a so-called quiescent condition of DTPs allows them to survive for long periods of time (weeks to months) in the time frame between being killed and developing mutations.13 This window of opportunity seems essential for DTPs—or at least parts of DTPs, to acquire mutation-driven resistance mechanisms by which they can evolve into clinically relevant drug-resistant cells.23,24

As such, the tolerance/dormancy/persistence state, which is accepted as an alternative route for acquiring resistance, tends to serve as a “bridge” to link the non-mutational mechanisms with bona fide resistance mechanisms (i.e., to connect phenotype-dependent DTPs with genotype-dependent resistant cells24,25) (Fig. ​(Fig.11).

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Fig. 1
The genesis of DTPs according to natural selection theory (classical Darwinian selection), the Lamarckian induction concept, and the coexisting model. a The natural selection theory shows that the preexisting DTPs, here represented by CSCs, can be selected and enriched upon drug exposure. b The concept of Lamarckian induction attaches importance to the natural aptitude of tumor cells in adapting to pharmacologic interventions through different levels of epigenetic modifications, giving rise to the emergence and coexistence of DTPs in varying tolerant states. c The coexisting model suggests the dynamic transcriptional fluctuation at a single-cell level of resistance-related markers (“transcriptional noise”). A small fraction of tumor cells, whose expression of these resistance-related genes exceeds a certain threshold at the moment of treatment, can survive and be selected (the blue and yellow dot), marking a return to classical Darwinian selection. However, with increasing duration of drug exposure, such a stochastic, transient, fluctuated “survival mode” arrives at drug-refractory state through epigenetic modifications, ultimately resulting in the establishment of a DTP pool. These alterations in the epigenome, which can be summed up as “acquired inertia,” are in agreement with the concept of Lamarckian induction. The solid line represents the changes of resistance-related markers expression with treatment, while the dotted line represents those without treatment (below). CSC cancer stem cell, DTPs drug-tolerant persisters

Despite knowing the significant contributions made by DTPs to both non-mutational and mutational processes during resistance, controversies still exist concerning the genesis of DTPs between the natural selection theory (classical Darwinian selection), Lamarckian induction concept, and the coexisting model, as described below26 (Fig. ​(Fig.1).1).

The natural selection theory is a simple and intuitive principle. Specifically, DTPs, here represented by CSCs in an inconspicuous but preexisting form which are hidden by the overwhelming number of non-CSCs, can be selected and enriched upon drug exposure17,27 (Fig. ​(Fig.1a).1a).

This theory, based on phenotypic behavior, can also be interpreted as a process for selecting the pre-existing slow-cycling cells under treatment, for example, pre-existing JARID1B-expressing melanoma cells or ZEB2-expressing colorectal cancer cells.28,29

In contrast to the “passive” mode of Darwinian selection, the concept of Lamarckian induction attaches importance to the natural aptitude of tumor cells in adapting to internal or external stimuli actively, especially in response to pharmacologic interventions, essentially a concept of therapy-triggered “adaptation” (Fig. ​(Fig.1b).1b).

This adaptation, rather than the “one mutation, one outcome” dualistic model, is predominantly reflected in the dynamic change of a number of resistance-related genes through epigenetic events during treatment, laying a mechanistic foundation for the emergence and coexistence of DTPs in varying tolerant states26,30 (Fig. ​(Fig.1b).1b).

Among the resistance-related markers, the well-characterized drug efflux pump—multidrug resistance protein-1 (MDR1), serves as an example.31 In this case, a quick and robust response to vincristine exposure manifesting as phenotypic switching from a low- to high-efflux state, has been observed, which is proved to be a direct consequence of “active” MDR1 induction via single-cell longitudinal surveillance.31

More importantly, once such an induction is triggered, transcriptomic alterations tend to persist for a relatively long time after drug withdrawal31 termed “epigenetic memory.”32 This is in accordance with the notion that DTPs can transiently evade treatment and maintain the pro-survival phenotype or transcriptome alterations for some time.12,26

In actual fact, the dynamic transcriptional fluctuation of resistance-related markers at a single-cell level is more likely to occur before the addition of drug in a manner similar to the so-called “transcriptional noise,” thus giving rise to an incremental source of transcriptional variability for drug selection16,32–35 (Fig. ​(Fig.1c).1c).

As a result, a small fraction of tumor cells, whose expression of these resistance-related genes exceeds a certain threshold at the moment of treatment, can survive or be selected.16 The “internal noise” (e.g., random pattern of transcriptional variability on resistance-related genes) can be viewed as a loaded “weapon” within the “arsenal” of tumor cells to cope with “external noise”36 (e.g., drug exposure), marking a return to classical Darwinian selection (Fig. ​(Fig.1c).1c).

However, with increasing duration of drug exposure, such a stochastic, transient, fluctuated “survival mode” develops into an adaptive, stable, dormant, drug-refractory state through epigenetic modifications, ultimately resulting in the establishment of a pool of DTPs.16,34

These alterations in the epigenome (i.e., “adapting to shape change instead of being shaped”) are in agreement with the concept of Lamarckian induction31 (Fig. ​(Fig.1c).1c). Hence, throughout the entire process of the emergence and maintenance of DTPs, these two concepts are not opposite, but rather intertwined and complementary to each other (Fig. ​(Fig.1c1c).

If one regards the profound transcriptional variability16 as the “innate skill” of tumor cells to pursue greater phenotypic diversity, the epigenome-associated dormant state caused by long-term treatment will be more likely the “acquired inertia” of DTPs due to the assumption that the survival skills, that is, overexpression of resistance-related genes, have been gained from the cells surviving initial therapy.

This raises the question of “when treatment is discontinued, will the ‘acquired inertia’ fade away and/or ‘innate ability’ be restored?” Consistent with in vitro laboratory experiments, the so-called ‘re-treatment response’’ phenomenon observed clinically supports the occurrence of a reversible process from acquired drug-refractory to initial drug-susceptible state following drug withdrawal37 (Fig. ​(Fig.1c).1c).

Specifically, a significant fraction of patients with NSCLC who have been through a failed treatment with epidermal growth factor receptor (EGFR)-TKI-based therapy (gefitinib) can immediately achieve remarkable tumor regression following re-treatment with gefitinib after a drug-free interval, demonstrating a second response “window” to treatment with TKIs.37,38

Similar re-treatment responses in different cancer types have also been observed with other anticancer agents, including daratumumab,39 trastuzumab,40 radium-223,41 and pembrolizumab.42,43 The prerequisite for such a secondary response is that the timing of re-treatment needs to precede the presence of a novel resistance-conferring mutation in DTPs. This can be interpreted as a process of residual DTPs getting rid of the “acquired inertia” while re-activating the “innate skill” or, put another way, a transition from a slow-cycling, drug-refractory to a fast-cycling, drug-susceptible phenotype (Fig. ​(Fig.1c1c).

Indeed, this reversible phenotype switching, at first glance, can be attributed to the proactive behavioral “changes” of tumor cells to adapt to environmental “changes” albeit in an uncontrollable manner. This also implies that hijacking the mechanisms underlying these “changes” for therapeutic purposes, transforming such a process from uncontrolled to controlled, could be a promising approach. For this reason, studies revolving around the complicated cellular mechanisms involved in the “war” of “hide (phenotype switching)-and-seek (cancer therapy)44” have gained increasing prominence in recent years.

In terms of phenotype switching, cell plasticity (the fundamental ability of cells to change their properties in a reversible way actively or passively) plays a prominent role in postinjury tissue repair and regeneration, as well as the restoration of disrupted homeostasis.45–47

Besides making contributions to such physiological processes, when activated aberrantly, cell plasticity is involved in the evolution and progression of multiple diseases, particularly cancer.46–49 This sheds new light on the explanation of the intratumoral heterogeneity of phenotypic features of cancer during which tumor cells exhibit varying degrees of phenotypic interconversion between drug-susceptible and drug-refractory states.50

The above general description of phenotype switching in cases of drug exposure or drug withdrawal represents a universally applicable model of tumor cell plasticity, regardless of what types of cancer are treated or what kind of therapies are employed. However, behind this universally plastic behavior, there exist differences in exactly how cancer cells evade therapy including epithelial–mesenchymal transition (EMT), acquiring properties of CSCs or trans-differentiation potential26,47,51–54 (Fig. ​(Fig.1c).1c).

Intriguingly, these somewhat functionally overlapping processes are more or less associated with the aberrant (re-)activation of developmental programs, suggesting that similar molecular mechanisms underlying plasticity-driven resistance to therapy may be involved.55,56

In summary, in this review, we present a comprehensive description of tumor cell plasticity in response to treatment of various cancers with respect to targeted therapies, chemotherapy, and immunotherapy, and will highlight the mechanisms involved.

Epithelial–mesenchymal transition (EMT)
The programs of EMT and its inverse process, mesenchymal-to-epithelial transition (MET), are involved in governing vertebrate embryonic development in a highly dynamic, transitory and reversible manner, representing a prime example of cell plasticity, both in normal and neoplastic cells.55,57,58

At conceptual and morphological levels, cells undergoing EMT are characterized by loss of apical–basal polarity and the disruption of cell–cell contacts, including tight (e.g., ZO-1), adherens (e.g., E­-cadherin), and gap junctions (e.g., connexins), while acquiring the front–rear polarity and dramatic remodeling of the cytoskeleton organization. This ultimately results in the morphotype switching from “cobblestone-like” shapes to “fibroblast-like” (e.g., vimentin) forms.55,59–61

Mechanistically, this process is generally performed by several EMT‑inducing transcription factors (EMT-TFs), such as Snail, zinc-finger E-box-binding (Zeb), and basic helix–loop–helix TFs, and noncoding microRNAs (miRNAs), epigenetic, and post-translational regulators, as well as alterative splicing factors, which are further integrated and controlled by multiple signaling pathways, such as the transforming growth factor-β (TGF-β), wingless/integrated (Wnt), Notch, and Ras-mitogen-activated protein kinase (Ras-MAPK) pathways, in response to paracrine and autocrine stimuli62–64 (Fig. ​(Fig.3a).3a).

Notably, the EMT-TFs are orchestrated and dynamically regulated themselves by each other and/or other factors in every step of EMT programming, in particular, the two well-established double-negative feedback loops, miR-34/Snail1 and miR-200/Zeb (Fig. ​(Fig.3a).3a).

The former regulatory circuit preferentially participates in the initial phase of EMT induction in epithelial cells, while the latter tends to be involved in the development and maintenance of the mesenchymal state.65–71 Functionally, it is generally recognized that the EMT programs not only play an irreplaceable role in multiple physiological processes throughout the whole course of an individual’s life, especially during embryonic development (tissue morphogenesis and organogenesis), wound healing, tissue repair, and the induction of pluripotency, but also contribute to various pathological events, including formation of fibrosis and tumor malignancy—from its genesis to development.59,72–75

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Fig. 3
The role of EMT in EGFR-TKI resistance. a Cancer cells undergoing EMT are characterized by morphotypic switching from “cobblestone-like” shapes to “fibroblast-like” forms. This process can be achieved via several EMT-TFs (Snail, Zeb, and Twist) and miRNAs in response to paracrine and autocrine stimuli, endowing cancer cell with a more aggressive phenotype, including enhanced invasive capacity, therapeutic resistance (enhanced drug efflux and slow cell proliferation), and stemness properties. b In EGFR-mutant NSCLC, upregulation of TEAD-mediated YAP promotes the transcription of Slug, which further induces the upregulation of AXL in NSCLC cells. AXL signaling, whose activation relies on interactions with its specific ligand GAS6, promotes EMT that drives Slug-overexpressing mesenchymal cells to acquire resistance with erlotinib. In addition, the mesenchymal cells display enhanced resistance to EGF816 accompanied by a significant activation of the FGFR1 pathway, implicating the potential of FGFR1 as a drug target for evading resistance to EGF816. A subpopulation of cancer cells can enter a senescence-like state to escape cell death upon administration of EGFRi (osimertinib) in combination with MEKi (tretinamib), resulting in resistance. This change is characterized by YAP/TEAD-mediated activation of EMT programs. The therapeutic strategy of pharmacologically cotargeting YAP/TEAD (by MYF-01-37) and EGFR/MEK leads to synthetic lethality. AXL anexelekto, GAS6 growth arrest-specific protein 6, SGI-7079/XL-880 AXL inhibitor, EGF816 the third-generation EGFR-TKIs, FGFR1 fibroblast growth factor receptor 1, BGJ398: FGFR inhibitor

. . . . . .

For cancer cells: better to change than be killed

At first sight, accompanied by the development of emerging therapeutic strategies (e.g., targeted therapy and immunotherapy), coupled with a solid understanding of the genetic mutations involved, advanced or even chemo-/ radiation-resistant cancers seem to be curable clinically.

However, the facts suggest otherwise. While initial clinical responses to patients with later-stage carcinomas typically appear encouraging, tumor recurrence inevitably occurs in these patients after a short-lived period of non-progression. This can be evidenced by the development of molecularly targeted therapies, i.e., three generations of EGFR-TKIs, to treat EGFR-mutant NSCLC, the results of which still have not been able to meet clinical expectations due to the acquisition of resistance. What then is the cause of this phenomenon?

It could be interpreted as a consequence of de novo mutations, or similar mechanisms, which endow tumor cells with the capability of bypassing inhibition of the targeted pathway under drug exposure. However, these explanations from the perspective of genetic alterations do not fully account for the accumulating clinical and laboratory observations, thus leading to a shift in research priority, at least in part, from mutational mechanisms to those related to non-genetic alterations.

The non-mutational process largely depends on tumor cell plasticity, which is regulated by highly integrated and complex interactions between transcriptional factors, epigenetic modulators as well as a variety of growth factors, cytokines, and chemokines released from non-neoplastic cells within the TME.

The impressive ability of tumor cells to switch their identities or phenotypes is more likely a common mechanism by which they can escape treatment. It should be noted that phenotypic “change” is often accompanied by the acquisition of a more aggressive behavior, especially enhanced flexibility, mainly manifested in the processes such as EMT, transition from non-CSC to CSC, or CRPC to NEPC, which will exacerbate the difficulty of clinical treatment.

Even more surprising, in most cases, tumor cells can achieve a new phenotype without losing their original properties, suggesting that phenotype switching between two functionally independent states is not strictly adhering to a binary-based “all or nothing” principle, but rather is a complicated multistage dynamic process involving several intermediate phenotypes with varying degrees of maintained biological characteristics.

Alternatively, plasticity may have already existed in the “arsenal” of tumor cells prior to drug exposure and thus cancer therapy actually serves as a “trigger” to stimulate “change” to avoid cell death—better to change than be killed. Although tumor plasticity has been proven to play a key role in resistance to cancer therapy, there remain numerous questions to be answered and challenges to face.

For treatment: only “change” can prevent “change”, and make it changeless
Given its malleable nature and consequent poor clinical outcomes, understanding the true meaning of plasticity (“change”) is fundamental to unlocking the secrets of non-mutational resistance mechanisms during cancer therapy. To deal better with the “change” of carcinoma cells, it will be necessary to change both experimental methods and treatment strategies.

Using the example of EMT described earlier in this review, the cognitive evolution of the EMT concept from a “complete” to a “partial” form, to a great extent, could be viewed as a reflection of the development of experimental techniques (i.e., from dual-colorimetric RNA-ISH to scRNA-seq to LSR-3D imaging).

This suggests that the ideal approach would monitor the whole dynamic process of cancer development from one phenotype to another, at both an individual and multicellular cluster level. Only when the nature of tumor plasticity is fully understood can complete prevention be truly achieved. This is likely to be based on not only existing strategies, such as intermittent treatment and combination therapy, but also the development of new strategies, such as adipogenesis therapy, which can take advantage of the vulnerability of tumor plasticity.

Finally, knowing that tumor cell plasticity plays an important role in therapeutic resistance, the prevention of this dynamic process seems to be a necessary prerequisite for the improvement of clinical outcomes for cancer patients. This assumes that the “change” of experimental methods is conducive to increasing our understanding of the mechanisms of the phenotypic “change” in cancer cells during treatment, which in turn could accelerate the “change” of therapeutic strategies to prevent tumor cell plasticity. In essence, only “change” can prevent “change,” and make it changeless.

reference link:

More information: Cihangir Duy et al. Chemotherapy induces senescence-like resilient cells capable of initiating AML recurrence, Cancer Discovery (2021). DOI: 10.1158/2159-8290.CD-20-1375


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