Mitochondria and chronic effects of cancer therapeutics the clinical implications

Abstract

One of the major mechanisms of action of chemo-radiation is to induce cellular senescence, which exerts crucial roles in age-related pathology. The concept of senescence is evolved, and the novel understanding of senescence-associated reprogramming/stemness has emerged. This new concept emphasizes senescence as not only cell cycle arrest but describes that subsets of senescent cells induced by chemotherapy can re-enter cell cycles, proliferate rapidly, and acquire “stemness” status. Cancer therapeutics, including chemo-radiation triggers toxicity effects through damaging mitochondria, primarily through the upregulation of mtROS production leading to subsequent mtDNA and telomeric DNA damage elicitng DNA damage responses (DDR). . The ultimate goal of this review is to highlight the new concept of senescence-associated stemnessthat is induced by cancer treatment and its adverse effects on the vascular system. . We will describe how chemo-radiation exerts toxicity effects by simultaneously producing reactive oxygen species in mitochondria and promoting DDR in the nucleus. We discuss the potential of clinical targeting poly (ADP-ribose) polymerase which might prevent downstream mitochondrial dysfunction and confer protection to cancer survivors. Overall we emphasize the importance of recognizing the consequences of cardio-toxic effects of several cancer treatments and therefore developing personalized therapeutic approaches to screen for inflammatory and cardiac testing for better patient survival.

Introduction:

Cardiotoxicity is an important complication of cancer therapies¹. The relationship of cancer survivorship to cardio-vascular diseases (CVD) is well documented: patients have 2–6 times higher CVD mortality than non-cancer general population with the majority in patients diagnosed with breast, prostate or bladder cancer². The increased risk is associated with a variety of therapies including radiation³. The CVD include not only cardiomyopathy but atherosclerosis, valvular and pericardial related morbidities.

It is necessary to emphasize that not only established therapies such as anthracyclines, antimetabolites, and alkylating agents but also modern therapies such as tyrosine kinase inhibitors (TKIs), angiogenesis inhibitors and checkpoint inhibitors induce cardiovascular (CV) toxicity¹. This is apparent from the study carried out by Lipshultz et al., where they compared two groups of cancer survivors, where one group was exposed to cardiotoxic drugs and the other group was not exposed to such treatment, and intriguingly authors found that both groups exhibited high levels of mean body mass index, fasting serum non-high-density-lipoprotein, insulin, high sensitivity c-reactive protein, and CV toxicity effects. This finding indicated that all cancer survivors were at high CV risk regardless of their cardiotoxic treatment exposure⁴, highlighting that although certain treatments are classified as non-cardiotoxic they have significant effect on the vasculature, specifically on vascular endothelial cells (ECs)^{5, 6}. It is critical to understand that even non-cardiotoxic therapeutic regiments induce pre-mature aging or accelerated senescence phenotypes, which is the underlying cause for CV toxicity observed in patients^{4, 6–11}.

One of the primary mechanisms through which cancer treatments induce toxic events is by induction of a cellular state called replicative senescence. Replicative senescence is the ceased proliferation of cells over time, ultimately leads to an irreversible cell cycle arrest and is characterized by accelerated telomere (TL) attrition/erosion, DNA damage, chromosomal aberrations, and chromatin condensation, etc^{6, 12}. During senescence TL attrition is not only through TL shortening but, also caused by significant oxidative DNA damage. Notably, DNA damage foci are found at TL regions in cancer therapy induced senescence independent of TL length and TL activity¹³.

Senescence induced by chemo-radiation can be categorized as stress-induced pre-mature aging/senescence (SIPS) and is markedly different from time dependent pre-mature aging/senescence^{6, 14}. Unlike time dependent senescence, SIPS is characterized by excessive reactive oxygen species (ROS) production and ROS is the governing factor in several senescent phenotypes¹⁴. Subsets of such senescent cells evade cell cycle arrest and undergo cell-autonomous reprogramming to acquire “stemness” status. These senescent cells with gained “stemness” status exhibit enhanced proliferative potential accompanied by the production of ROS, inflammatory cytokines and growth factors, is termed as senescence-associated secretory phenotype (SASP). By inducing SASP, senescent cells promote inflammatory and proliferative signaling thus leading to aggressive proliferative phenotypes¹⁵. Therefore, although SASP shows typical senescence characterization such as TL dysfunction or the increase of senescence-associated β-gal staining, SASP is not necessarily related to cell cycle arrest and cell death^{15, 16}. Senescent cells with acquired stemness and SASP along with enhanced proliferative capacity, enables cancer therapy resistance, malignant conversion, tumor progression, as well as immune modulation and regeneration¹⁷.

Anti-cancer drugs induce mitochondrial ROS (mtROS) production by directly and indirectly affecting mitochondrial function:

Mitochondria produce energy for the cells through respiration (glycolysis, transition reaction, the Kreb’s cycle, and the mitochondrial OXPHOS, mtOXPHOS). The mtOXPHOS is composed of the mitochondrial electron transport chain (mtETC) and the ATP production, which include membrane-embedded proteins that are organized into five complexes (I-V). ROS are mainly generated in mitochondria via the mtETC. The majority of mtROS is produced by complexes I, II, and III through O₂ interacting with electrons derived from NADH and FADH₂. In brief, NADH and FADH₂ carry electrons to the mtETC, passing down an electrochemical gradient to be delivered to O₂ via a chain of respiratory proton pumps¹⁸. Because mitochondria regulate critical biological functions including energy production (mtOXPHOS or oxidative phosphorylation through mtETC), cell cycles (cell death and proliferation), cell metabolism, and mitochondrial-to-nuclear signaling, the dysfunction of mitochondria has a vital role in the pathology of many diseases including cancer therapy-related diseases. Mitochondrial dysfunction can be initiated by (i) dysfunction of the mtETC, the Krebs cycle, the mtOXPHOS, and the fatty acid β-oxidation; (ii) losing of mitochondrial membrane potential, and (iii) increasing of ROS production and decreasing of antioxidative capacity.

Several cancer therapeutic agents have adverse effects on mitochondria and cause severe mitochondrial damage, specifically through upregulation of mtROS, which is believed to be the critical step leading to CV toxicity¹⁹. Oxidative damaging and aging have been documented and reviewed^{20, 21}. Mitochondrial and cellular hydrogen peroxide (H₂O₂) form two highly reactive derivatives, hydroxide ion (OHˉ) and hydroxyl radical ˙OH via the Fenton reaction^{22, 23}. This reaction has been linked to aging senescence state with loss of stemness²⁴ and also to SIPS state in cancer¹². The increased mitochondrial ROS production in cancer results from mutated mtDNA and nDNA encoded electron transport chain complex structural changes resulting in reduced respiratory capacity and enhanced leak²⁵ For example, doxorubicin can specifically bind the phospholipid cardiolipin located on the mitochondrial inner membrane and accumulate in mitochondria²⁶. Mitochondrial accumulation of doxorubicin directly inhibits complex I and II, disrupting the mtETC and resulting in mtROS production²⁷. On the another hand, ROS-induced apoptosis has been viewed as a major mechanism that is activated by monoclonal antibodies and Tyrosine Kinsae Inhibitors²⁸. The monoclonal antibody rituximab activates ROS-induced apoptosis through abolishing Bcl-2 and p38MAPK signaling. TKIs such as imatinib, erlotinib, and vemurafenib induce mtROS production by disrupting mitochondrial membrane potential via activating cytosolic kinases of JNK and p38 indirectly^{28, 29}. It has also been reported that mitochondria is the major target during cisplatin treatment and it is believed to interfere with mitochondrial metabolic funtions³⁰. Taken together the link between cancer therapy, mitochondria and aging appears to be enhanced ROS production of different etiologies that induce downstream adverse health effects.

PARP: Different roles in cancer and CVD.

Poly (ADP-ribose) polymerase (PARP), a highly conserved nuclear enzyme, functions as a zinc-dependent DNA damage sensor that can bind to both single-stranded DNA breaks (SSBs) and double-stranded DNA breaks (DSBs)³¹. Via binding to DNA breaks, PARP is activated. PARP activation causes PARylation of nuclear proteins, also known as polyADP-ribosylation, by converting DNA damage into either DNA repair or cell death signaling pathways³². To determine PARP function in the regulation of DNA damage repair, PARP inhibitors have been developed. The inhibition of PARP is associated with the increased sensitivity to DNA-alkylating agents, enhances antitumor activity of DNA-methylating agent, topoisomerase poisons, and ionizing radiation. In two independent studies, the combination therapy including temozolomide and PARP inhibitor results in the complete tumor regression. For the first time, the combination of temozolomide and PARP inhibitor is undergoing clinical evaluation³².

When chemo-radiation causes SSBs, the DNA SSB recognition domain on PARP can recognize SSBs immediately, thus promoting the formation of a PARP1 homodimer at the site of damage and catalyzing nicotinamide adenine dinucleotide (NAD⁺) breakdown into ADP-ribose and nicotinamide. Recently, the critical role of PARP activation in the inhibition of both OXPHOS and glycolysis, as well as the reduction of NAD⁺, NADH, and ATP level following DNA damage, was reported³⁴ (Fig .1). NAD⁺ is an important mediator for several metabolic functions of mitochondria and reduction in NAD+ levels could lead to adverse effects³⁵.

Fig. 1. NAD⁺, NAMPT, and PARPs.

NAD⁺ is synthesized from nicotinamide (NAM) by NAMPT. NAD⁺ is broken down by PARPs and sirtuins (SIRTs). PARPs creates polymers of ADP ribose (ADRP) that are often attached to a protein substrate. SIRTS transfer an O-acetyl group from a protein substrate to the ADPR moiety of NAD⁺ to yield O-acetyl-ADPR and Nam. Activation of PARPs consume NAD⁺, and inhibit SIRT1 activity by limiting NAD⁺ level and by PARylation of DNA and proteins.

Specifically, NAD⁺ depletion inactivates sirtuins (SIRT1) deacetylase activity, leading to mitochondrial dysfunction. Also, the contribution of pathophysiological reduction of NAD⁺ synthesis and subsequent SIRTs activity during the aging process has been suggested³⁶. The reduction of SIRT activity induced by NAD⁺ depletion can provoke mitochondrial dysfunction by several different mechanisms; 1) the reduction of SIRT1 activity inhibits mitochondrial transcription factor TFAM by stabilizing HIF-1α and subsequent inhibition of c-Myc. The down-regulation of TFAM expression evokes mitochondrial metabolic dysfunction by reducing mitochondrial gene expression^36–38, 2) PGC-1α and FOXO1 function was inhibited by the reduction of SIRT1 activity³⁹, attenuating mitochondrial biogenesis, oxidative metabolism, and anti-oxidant defense pathways, 3) PGC-1α, through co-activation of ERR and PPAR, can upregulate components of mitophagy and dynamics⁴⁰. Therefore, the reduction of SIRT1 activity can increase mtROS production by inhibiting oxidative metabolism, anti-oxidant defense pathway, and also by down-regulating mitophagy which leads to accumulation of damaged mitochondria^{41, 42}.

H₂O₂ generated by mitochondria can be diffused in the nucleus, and elicit TL DNA damage but not genomic DNA damage and activating DNA damage response (DDR)⁴³. These data indicate the crucial role of TL but not genomic DNA damage in mtROS-induced PARP activation (one of the DDR components), and subsequent NAD⁺ depletion and mitochondrial dysfunction as we explained above (Fig .2.). These data suggest that there is a crosstalk between the nucleus and mitochondria, which is regulated by mitochondrial-to-nuclear signaling pathways that cause sustained mtROS production and leading to pre-mature aging via TL DNA damage by forming positive feedback loop (Fig .2.).

Fig. 2. The crosstalk between mitochondria and nucleus mediated by mtROS-induced telomere (TL) DNA damage and subsequent NAD⁺ depletion.

Mt: mitochondria, PARP: poly (ADP-Ribose) polymerase, TFAM: transcription factor A, mitochondria, SIRT1: sirtuin 1, PGC-1α Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-α FOXO: Forkhead Box O1. (made by bioRENDER)

In non-oncological tissue such as ECs (low PARP expression with intact homologous recombination), PARP inhibition can be cytoprotective and anti-inflammatory by preventing NAD⁺ depletion and eventually preventing CVD (Figs. 1 and 4). Re-purposing PARP inhibitors (anti-tumor agents) to prevent IR-induced CVD in cancer patients would be an ideal therapeutic intervention, but to our best knowledge, no studies of this approach, including pre-clinical studies for preventing the RT-induced late effects of CVD, have been reported.

Clinical considerations/impact:

As example of the clinical impact of this one might consider cancer survivors risk of CVD. A 2019 report by Sturgeon, K.M. et al documented that although cancer survivorship has increased, greater than 10% will die prematurely from cardiovascular disease and that the risk of death was highest in the first year after the cancer diagnosis². Increasing awareness of the underlying mechanisms could lead to early recognition and precision medical treatment. Heart failure and atherosclerosis are predominant causes of mortality. Therefore, laboratory screening for inflammation, thrombotic risk, hyperlipidemia, metabolic syndrome, diabetes along with blood pressure control and cardiac testing to include monitoring of left ventricular systolic and diastolic function, surveillance cardiac imaging, etc. Cardio-oncology as a sub-specialty to address this has emerged.

Concluding remarks:

The late effects after chemo-radiation are evidenced; however, this subject has been dominated by the acute effects of cancer therapy in cardiovascular diseases. Nevertheless, the accelerated pre-mature aging is essential not only in young cancer survivors but also in the elderly or patients with pre-existing CVDs who exhibit poor clinical outcomes following cancer therapy. As such, it is critical to understand mechanisms that cause accelerating pre-mature aging in cancer survivors at the molecular level. Because pre-mature aging differs from cell cycle arrest, it is reversible and, therefore, can be an ideal target for therapeutic intervention. Importantly, the concept of senescence-associated reprogramming or stemness, and its potential role in cancer therapy-mediated SASP induction and its downstream clinical phenotypes of cardio-toxic effects requires further study. . These insights will better equip clinicians to design personalized cancer therapeutic approaches and improve patient survival. In this review, we discuss the potential of targeting PARP, which prevents subsequent mitochondrial dysfunction and senescence induction. We strongly believe that future studies will address and develop novel strategies to prevent senescence induction prior to cancer treatment.