Changing the Landscape of Solid Tumor Therapy from Apoptosis-Promoting to Apoptosis-Inhibiting Strategies.

The many limitations of implementing anticancer strategies under the term "precision oncology" have been extensively discussed. While some authors propose promising future directions, others are less optimistic and use phrases such as illusion, hype, and false hypotheses. The reality is revealed by practicing clinicians and cancer patients in various online publications, one of which has stated that "in the quest for the next cancer cure, few researchers bother to look back at the graveyard of failed medicines to figure out what went wrong". The message is clear: Novel therapeutic strategies with catchy names (e.g., synthetic "lethality") have not fulfilled their promises despite decades of extensive research and clinical trials. The main purpose of this review is to discuss key challenges in solid tumor therapy that surprisingly continue to be overlooked by the Nomenclature Committee on Cell Death (NCCD) and numerous other authors. These challenges include: The impact of chemotherapy-induced genome chaos (e.g., multinucleation) on resistance and relapse, oncogenic function of caspase 3, cancer cell anastasis (recovery from late stages of apoptosis), and pitfalls of ubiquitously used preclinical chemosensitivity assays (e.g., cell "viability" and tumor growth delay studies in live animals) that score such pro-survival responses as "lethal" events. The studies outlined herein underscore the need for new directions in the management of solid tumors.

Single-Cell MTT: A Simple and Sensitive Assay for Determining the Viability and Metabolic Activity of Polyploid Giant Cancer Cells (PGCCs).

Solid tumors and tumor-derived cell lines commonly contain highly enlarged (giant) cancer cells that enter a state of transient dormancy (active sleep) after they are formed, but retain viability, secrete growth promoting factors, and exhibit the ability to generate rapidly proliferating progeny with stem cell-like properties. Giant cells with a highly enlarged nucleus or multiple nuclei are often called polyploid giant cancer cells (PGCCs). Although PGCCs constitute only a subset of cells within a solid tumor/tumor-derived cell line, their frequency can increase markedly following exposure to ionizing radiation or chemotherapeutic drugs. In this chapter we outline a simple and yet highly sensitive cell-based assay, called single-cell MTT, that we have optimized for determining the viability and metabolic activity of PGCCs before and after exposure to anticancer agents. The assay measures the ability of individual PGCCs to convert the MTT tetrazolium salt to its water insoluble formazan metabolite. In addition to evaluating PGCCs, this assay is also a powerful tool for determining the viability and metabolic activity of cancer cells undergoing premature senescence following treatment with anticancer agents, as well as for distinguishing dead cancer cells and dying cells (e.g., exhibiting features of apoptosis, ferroptosis, etc.) that have the potential to resume proliferation through a process called anastasis.

Intratumor Heterogeneity and Treatment Resistance of Solid Tumors with a Focus on Polyploid/Senescent Giant Cancer Cells (PGCCs).

Single cell biology has revealed that solid tumors and tumor-derived cell lines typically contain subpopulations of cancer cells that are readily distinguishable from the bulk of cancer cells by virtue of their enormous size. Such cells with a highly enlarged nucleus, multiple nuclei, and/or multiple micronuclei are often referred to as polyploid giant cancer cells (PGCCs), and may exhibit features of senescence. PGCCs may enter a dormant phase (active sleep) after they are formed, but a subset remain viable, secrete growth promoting factors, and can give rise to therapy resistant and tumor repopulating progeny. Here we will briefly discuss the prevalence and prognostic value of PGCCs across different cancer types, the current understanding of the mechanisms of their formation and fate, and possible reasons why these tumor repopulating "monsters" continue to be ignored in most cancer therapy-related preclinical studies. In addition to PGCCs, other subpopulations of cancer cells within a solid tumor (such as oncogenic caspase 3-activated cancer cells and drug-tolerant persister cancer cells) can also contribute to therapy resistance and pose major challenges to the delivery of cancer therapy.

The Cellular Response to DNA Damage: From DNA Repair to Polyploidy and Beyond.

A major challenge in treating patients with solid tumors is posed by intratumor heterogeneity, with different sub-populations of cancer cells within the same tumor exhibiting therapy resistance through different biological processes [...].

What Are the Reasons for Continuing Failures in Cancer Therapy? Are Misleading/Inappropriate Preclinical Assays to Be Blamed? Might Some Modern Therapies Cause More Harm than Benefit?

Over 50 years of cancer research has resulted in the generation of massive amounts of information, but relatively little progress has been made in the treatment of patients with solid tumors, except for extending their survival for a few months at best. Here, we will briefly discuss some of the reasons for this failure, focusing on the limitations and sometimes misunderstanding of the clinical relevance of preclinical assays that are widely used to identify novel anticancer drugs and treatment strategies (e.g., "synthetic lethality"). These include colony formation, apoptosis (e.g., caspase-3 activation), immunoblotting, and high-content multiwell plate cell-based assays, as well as tumor growth studies in animal models. A major limitation is that such assays are rarely designed to recapitulate the tumor repopulating properties associated with therapy-induced cancer cell dormancy (durable proliferation arrest) reflecting, for example, premature senescence, polyploidy and/or multinucleation. Furthermore, pro-survival properties of apoptotic cancer cells through phoenix rising, failed apoptosis, and/or anastasis (return from the brink of death), as well as cancer immunoediting and the impact of therapeutic agents on interactions between cancer and immune cells are often overlooked in preclinical studies. A brief review of the history of cancer research makes one wonder if modern strategies for treating patients with solid tumors may sometimes cause more harm than benefit.

Nonlinearities in the cellular response to ionizing radiation and the role of p53 therein.

Many aspects of the cellular response to agents such as ionizing radiation that cause genotoxic and/or oxidative stress exhibit a nonlinear relationship to the applied stress level. These include elements of the antioxidant response and of the damage-signaling pathways that determine cell fate decisions. The wild-type p53 protein, which is mutated in many cancers, coordinates these responses and is a key determinant of this nonlinearity. Indeed, p53 has been referred to as a 'cellular rheostat' that favors antioxidant/cytoprotective functions at low stress levels while switching to a pro-oxidant/cytotoxic role under high-stress conditions. For solid tumor-derived cell lines, moderate doses of radiation, typical of those used to generate clonogenic survival curves (i.e. ≤10 Gy), predominantly invoke a dose-dependent cytostatic response. For cancer cell lines with wild-type p53, cytostasis is primarily associated with features of senescence, whereas cancer cells with aberrant p53 primarily undergo endopolyploidization and enlargement. In line with a commentary by Meyn et al. [Int J Radiat Biol. 2009, 85:107-115] concluding that apoptosis is not the primary cause of radiation-induced loss of clonogenicity in solid tumor-derived cell lines, significant levels of apoptosis are typically seen only after higher doses (≥5 Gy) and this is almost all of the delayed (rather than primary) type. Nonlinearity of the oxidative/genotoxic stress response is already apparent in the early antioxidant events activated by transcription factors such as p53 and Nrf2 and the Ref1 transcription coactivator. These cytoprotective pathways serve to minimize damage to important cellular targets caused by reactive oxygen species (ROS) and other electrophiles. After high/supra-lethal levels of stress these inducible antioxidant pathways can be deactivated in a manner that would reinforce the establishment of the pro-oxidant state, resulting in elevated ROS levels and to cytostasis or apoptosis. Understanding the complex regulation of these damage-signaling pathways in relation to the stress levels is important for the optimal utilization of radiation therapy for cancer.

Do TUNEL and Other Apoptosis Assays Detect Cell Death in Preclinical Studies?

The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay detects DNA breakage by labeling the free 3'-hydroxyl termini. Given that genomic DNA breaks arise during early and late stages of apoptosis, TUNEL staining continues to be widely used as a measure of apoptotic cell death. The advantages of the assay include its relative ease of performance and the broad availability of TUNEL assay kits for various applications, such as single-cell analysis of apoptosis in cell cultures and tissue samples. However, as briefly discussed herein, aside from some concerns relating to the specificity of the TUNEL assay itself, it was demonstrated some twenty years ago that the early stages of apoptosis, detected by TUNEL, can be reversed. More recently, compelling evidence from different biological systems has revealed that cells can recover from even late stage apoptosis through a process called anastasis. Specifically, such recovery has been observed in cells exhibiting caspase activation, genomic DNA breakage, phosphatidylserine externalization, and formation of apoptotic bodies. Furthermore, there is solid evidence demonstrating that apoptotic cells can promote neighboring tumor cell repopulation (e.g., through caspase-3-mediated secretion of prostaglandin E2) and confer resistance to anticancer therapy. Accordingly, caution should be exercised in the interpretation of results obtained by the TUNEL and other apoptosis assays (e.g., caspase activation) in terms of apoptotic cell demise.

Cellular Responses to Platinum-Based Anticancer Drugs and UVC: Role of p53 and Implications for Cancer Therapy.

Chemotherapy is intended to induce cancer cell death through apoptosis and other avenues. Unfortunately, as discussed in this article, moderate doses of genotoxic drugs such as cisplatin typical of those achieved in the clinic often invoke a cytostatic/dormancy rather than cytotoxic/apoptosis response in solid tumour-derived cell lines. This is commonly manifested by an extended apoptotic threshold, with extensive apoptosis only being seen after very high/supralethal doses of such agents. The dormancy response can be associated with senescence-like features, polyploidy and/or multinucleation, depending in part on the p53 status of the cells. In most solid tumour-derived cells, dormancy represents a long-term survival mechanism, ultimately contributing to disease recurrence. This review highlights the nonlinearity of key aspects of the molecular and cellular responses to bulky DNA lesions in human cells treated with chemotherapeutic drugs (e.g., cisplatin) or ultraviolet light-C (a widely used tool for unraveling details of the DNA damage-response) as a function of the level of genotoxic stress. Such data highlight the growing realization that targeting dormant cancer cells, which frequently emerge following conventional anticancer treatments, may represent a novel strategy to prevent or, at least, significantly suppress cancer recurrence.

Intratumor Heterogeneity and Therapy Resistance: Contributions of Dormancy, Apoptosis Reversal (Anastasis) and Cell Fusion to Disease Recurrence.

A major challenge in treating cancer is posed by intratumor heterogeneity, with different sub-populations of cancer cells within the same tumor exhibiting therapy resistance through different biological processes. These include therapy-induced dormancy (durable proliferation arrest through, e.g., polyploidy, multinucleation, or senescence), apoptosis reversal (anastasis), and cell fusion. Unfortunately, such responses are often overlooked or misinterpreted as "death" in commonly used preclinical assays, including the in vitro colony-forming assay and multiwell plate "viability" or "cytotoxicity" assays. Although these assays predominantly determine the ability of a test agent to convert dangerous (proliferating) cancer cells to potentially even more dangerous (dormant) cancer cells, the results are often assumed to reflect loss of cancer cell viability (death). In this article we briefly discuss the dark sides of dormancy, apoptosis, and cell fusion in cancer therapy, and underscore the danger of relying on short-term preclinical assays that generate population-based data averaged over a large number of cells. Unveiling the molecular events that underlie intratumor heterogeneity together with more appropriate experimental design and data interpretation will hopefully lead to clinically relevant strategies for treating recurrent/metastatic disease, which remains a major global health issue despite extensive research over the past half century.

Viability Assessment Following Anticancer Treatment Requires Single-Cell Visualization.

A subset of cells within solid tumors become highly enlarged and enter a state of dormancy (sustained proliferation arrest) in response to anticancer treatment. Although dormant cancer cells might be scored as "dead" in conventional preclinical assays, they remain viable, secrete growth-promoting factors, and can give rise to progeny with stem cell-like properties. Furthermore, cancer cells exhibiting features of apoptosis (e.g., caspase-3 activation) following genotoxic stress can undergo a reversal process called anastasis and survive. Consistent with these observations, single-cell analysis of adherent cultures (solid tumor-derived cell lines with differing p53 status) has demonstrated that virtually all cells-irrespective of their size and morphology-that remain adherent to the culture dish for a long time (weeks) after treatment with anticancer agents exhibit the ability to metabolize 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- tetrazolium bromide (MTT). The purpose of this commentary is to briefly review these findings and discuss the significance of single-cell (versus population averaged) observation methods for assessment of cancer cell viability and metabolic activity.

Defenses against Pro-oxidant Forces - Maintenance of Cellular and Genomic Integrity and Longevity.

There has been enormous recent progress in understanding how human cells respond to oxidative stress, such as that caused by exposure to ionizing radiation. We have witnessed a significant deciphering of the events that underlie how antioxidant responses counter pro-oxidant damage to key biological targets in all cellular compartments, including the genome and mitochondria. These cytoprotective responses include: 1. The basal cellular repertoire of antioxidant capabilities and its supporting cast of facilitator enzymes; and 2. The inducible phase of the antioxidant response, notably that mediated by the Nrf2 transcription factor. There has also been frenetic progress in defining how reactive electrophilic species swamp existing protective mechanisms to augment DNA damage, events that are embodied in the cellular "DNA-damage response", including cell cycle checkpoint activation and DNA repair, which occur on a time scale of hours to days, as well as the implementation of cellular responses such as apoptosis, autophagy, senescence and reprograming that extend the time period of damage sensing and response into weeks, months and years. It has become apparent that, in addition to the initial oxidative insult, cells typically undergo further waves of secondary reactive oxygen/nitrogen species generation, DNA damage and signaling and that these may reemerge long after the initial events have subsided, probably being driven, at least in part, by persisting DNA damage. These reactive oxygen/nitrogen species are an integral part of the pathological consequences of radiation exposure and may persist across multiple cell divisions. Because of the pervasive nature of oxidative stress, a cell will manifest different responses in different subcellular compartments and to different levels of stress injury. Aspects of these compartmentalized responses can involve the same proteins (such as ATM, p53 and p21) but in different functional guises, e.g., in cytoplasmic versus nuclear responses or in early- versus late-phase events. Many of these responses involve gene activation and new protein synthesis as well as a plethora of post-translational modifications of both basal and induced response proteins. It is these responses that we focus on in this review.

Roles of Polyploid/Multinucleated Giant Cancer Cells in Metastasis and Disease Relapse Following Anticancer Treatment.

Tumors and tumor-derived cell lines contain polyploid giant cells with significantly elevated genomic content, often with multiple nuclei. The frequency of giant cells can increase markedly following anticancer treatment. Although giant cells enter a dormant phase and therefore do not form macroscopic colonies (aggregates of ≥50 cells) in the conventional in vitro colony formation assay, they remain viable and metabolically active. The purpose of this commentary is to underscore the potential importance of polyploid/multinucleated giant cells in metastasis and cancer recurrence following exposure to anticancer agents. We also discuss the possibility that most preclinical (cell-based and animal model) drug discovery approaches might not account for delayed responses that are associated with dormant giant cells.

Do Multiwell Plate High Throughput Assays Measure Loss of Cell Viability Following Exposure to Genotoxic Agents?

Cell-based assays in multiwell plates are widely used for radiosensitivity and chemosensitivity assessment with different mammalian cell types. Despite their relative ease of performance, such assays lack specificity as they do not distinguish between the cytostatic (reversible/sustained growth arrest) and cytotoxic (loss of viability) effects of genotoxic agents. We recently reported studies with solid tumor-derived cell lines demonstrating that radiosensitivity as measured by multiwell plate colorimetric (e.g., XTT) and fluorimetric (e.g., CellTiter-Blue) assays reflects growth arrest but not loss of viability. Herein we report similar observations with cancer cell lines expressing wild-type p53 (A549 lung carcinoma) or mutant p53 (MDA-MB-231 breast carcinoma) after treatment with the chemotherapeutic drug cisplatin. Importantly, we show that treatment of cancer cells with concentrations of cisplatin that result in 50% effect (i.e., IC50) in multiwell plate assays trigger the emergence of growth arrested cells that exhibit highly enlarged morphology, remain viable and adherent to the culture dish, and metabolize the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) to its formazan derivative. The emergence of markedly enlarged viable cells complicates the interpretation of chemosensitivity data obtained with multiwell plate high throughput assays. Relying solely on IC50 values could be misleading.

Impact of Premature Senescence on Radiosensitivity Measured by High Throughput Cell-Based Assays.

In most p53 wild-type human cell types, radiosensitivity evaluated by the colony formation assay predominantly reflects stress-induced premature senescence (SIPS) and not cell death (Int. J. Mol. Sci. 2017, 18, 928). SIPS is a growth-arrested state in which the cells acquire flattened and enlarged morphology, remain viable, secrete growth-promoting factors, and can give rise to tumor-repopulating progeny. The impact of SIPS on radiosensitivity measured by short-term assays remains largely unknown. We report that in four p53 wild-type human solid tumor-derived cell lines (HCT116, SKNSH, MCF7 and A172): (i) the conventional short-term growth inhibition assay (3 days post-irradiation) generates radiosensitivity data comparable to that measured by the laborious and time-consuming colony formation assay; (ii) radiation dose-response curves obtained by multiwell plate colorimetric/fluorimetric assays are markedly skewed towards radioresistance, presumably reflecting the emergence of highly enlarged, growth-arrested and viable cells; and (iii) radiation exposure (e.g., 8 Gy) does not trigger apoptosis or loss of viability over a period of 3 days post-irradiation. Irrespective of the cell-based assay employed, caution should be exercised to avoid misinterpreting radiosensitivity data in terms of loss of viability and, hence, cell death.

Significance of Wild-Type p53 Signaling in Suppressing Apoptosis in Response to Chemical Genotoxic Agents: Impact on Chemotherapy Outcome.

Our genomes are subject to potentially deleterious alterations resulting from endogenous sources (e.g., cellular metabolism, routine errors in DNA replication and recombination), exogenous sources (e.g., radiation, chemical agents), and medical diagnostic and treatment applications. Genome integrity and cellular homeostasis are maintained through an intricate network of pathways that serve to recognize the DNA damage, activate cell cycle checkpoints and facilitate DNA repair, or eliminate highly injured cells from the proliferating population. The wild-type p53 tumor suppressor and its downstream effector p21WAF1 (p21) are key regulators of these responses. Although extensively studied for its ability to control cell cycle progression, p21 has emerged as a multifunctional protein capable of downregulating p53, suppressing apoptosis, and orchestrating prolonged growth arrest through stress-induced premature senescence. Studies with solid tumors and solid tumor-derived cell lines have revealed that such growth-arrested cancer cells remain viable, secrete growth-promoting factors, and can give rise to progeny with stem-cell-like properties. This article provides an overview of the mechanisms by which p53 signaling suppresses apoptosis following genotoxic stress, facilitating repair of genomic injury under physiological conditions but having the potential to promote tumor regrowth in response to cancer chemotherapy.

Multinucleated Giant Cancer Cells Produced in Response to Ionizing Radiation Retain Viability and Replicate Their Genome.

Loss of wild-type p53 function is widely accepted to be permissive for the development of multinucleated giant cells. However, whether therapy-induced multinucleation is associated with cancer cell death or survival remains controversial. Herein, we demonstrate that exposure of p53-deficient or p21WAF1 (p21)-deficient solid tumor-derived cell lines to ionizing radiation (between 2 and 8 Gy) results in the development of multinucleated giant cells that remain adherent to the culture dish for long times post-irradiation. Somewhat surprisingly, single-cell observations revealed that virtually all multinucleated giant cells that remain adherent for the duration of the experiments (up to three weeks post-irradiation) retain viability and metabolize 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), and the majority (>60%) exhibit DNA synthesis. We further report that treatment of multinucleated giant cells with pharmacological activators of apoptosis (e.g., sodium salicylate) triggers their demise. Our observations reinforce the notion that radiation-induced multinucleation may reflect a survival mechanism for p53/p21-deficient cancer cells. With respect to evaluating radiosensitivity, our observations underscore the importance of single-cell experimental approaches (e.g., single-cell MTT) as the creation of viable multinucleated giant cells complicates the interpretation of the experimental data obtained by commonly-used multi-well plate colorimetric assays.

The Growing Complexity of Cancer Cell Response to DNA-Damaging Agents: Caspase 3 Mediates Cell Death or Survival?

It is widely stated that wild-type p53 either mediates the activation of cell cycle checkpoints to facilitate DNA repair and promote cell survival, or orchestrates apoptotic cell death following exposure to cancer therapeutic agents. This reigning paradigm has been challenged by numerous discoveries with different human cell types, including solid tumor-derived cell lines. Thus, activation of the p53 signaling pathway by ionizing radiation and other DNA-damaging agents hinders apoptosis and triggers growth arrest (e.g., through premature senescence) in some genetic backgrounds; such growth arrested cells remain viable, secrete growth-promoting factors, and give rise to progeny with stem cell-like properties. In addition, caspase 3, which is best known for its role in the execution phase of apoptosis, has been recently reported to facilitate (rather than suppress) DNA damage-induced genomic instability and carcinogenesis. This observation is consistent with an earlier report demonstrating that caspase 3 mediates secretion of the pro-survival factor prostaglandin E2, which in turn promotes enrichment of tumor repopulating cells. In this article, we review these and related discoveries and point out novel cancer therapeutic strategies. One of our objectives is to demonstrate the growing complexity of the DNA damage response beyond the conventional "repair and survive, or die" hypothesis.

Characterization of the apoptotic response induced by the cyanine dye D112: a potentially selective anti-cancer compound.

Chemotherapeutic drugs that are used in anti-cancer treatments often cause the death of both cancerous and noncancerous cells. This non-selective toxicity is the root cause of untoward side effects that limits the effectiveness of therapy. In order to improve chemotherapeutic options for cancer patients, there is a need to identify novel compounds with higher discrimination for cancer cells. In the past, methine dyes that increase the sensitivity of photographic emulsions have been investigated for anti-cancer properties. In the 1970's, Kodak Laboratories initiated a screen of approximately 7000 dye structural variants for selective toxicity. Among these, D112 was identified as a promising compound with elevated toxicity against a colon cancer cell line in comparison to a non-transformed cell line. Despite these results changing industry priorities led to a halt in further studies on D112. We decided to revive investigations on D112 and have further characterized D112-induced cellular toxicity. We identified that in response to D112 treatment, the T-cell leukemia cell line Jurkat showed caspase activation, mitochondrial depolarization, and phosphatidylserine externalization, all of which are hallmarks of apoptosis. Chemical inhibition of caspase enzymatic activity and blockade of the mitochondrial pathway through Bcl-2 expression inhibited D112-induced apoptosis. At lower concentrations, D112 induced growth arrest. To gain insight into the molecular mechanism of D112 induced mitochondrial dysfunction, we analyzed the intracellular localization of D112, and found that D112 associated with mitochondria. Interestingly, in the cell lines that we tested, D112 showed increased toxicity toward transformed versus non-transformed cells. Results from this work identify D112 as a potentially interesting molecule warranting further investigation.

Spontaneous γH2AX Foci in Human Solid Tumor-Derived Cell Lines in Relation to p21WAF1 and WIP1 Expression.

Phosphorylation of H2AX on Ser139 (γH2AX) after exposure to ionizing radiation produces nuclear foci that are detectable by immunofluorescence microscopy. These so-called γH2AX foci have been adopted as quantitative markers for DNA double-strand breaks. High numbers of spontaneous γH2AX foci have also been reported for some human solid tumor-derived cell lines, but the molecular mechanism(s) for this response remains elusive. Here we show that cancer cells (e.g., HCT116; MCF7) that constitutively express detectable levels of p21WAF1 (p21) exhibit low numbers of γH2AX foci (<3/nucleus), whereas p21 knockout cells (HCT116p21-/-) and constitutively low p21-expressing cells (e.g., MDA-MB-231) exhibit high numbers of foci (e.g., >50/nucleus), and that these foci are not associated with apoptosis. The majority (>95%) of cells within HCT116p21-/- and MDA-MB-231 cultures contain high levels of phosphorylated p53, which is localized in the nucleus. We further show an inverse relationship between γH2AX foci and nuclear accumulation of WIP1, an oncogenic phosphatase. Our studies suggest that: (i) p21 deficiency might provide a selective pressure for the emergence of apoptosis-resistant progeny exhibiting genomic instability, manifested as spontaneous γH2AX foci coupled with phosphorylation and nuclear accumulation of p53; and (ii) p21 might contribute to positive regulation of WIP1, resulting in dephosphorylation of γH2AX.