University of Groningen Cellular Stress in Aging and Cancer Sturmlechner, Ines

University of Groningen

Cellular Stress in Aging and Cancer

Sturmlechner, Ines

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10.33612/diss.170212168

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2021

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Sturmlechner, I. (2021). Cellular Stress in Aging and Cancer. University of Groningen.

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CHAPTER 1

General introduction

Homeostasis

“The living being is an agency of such sort that each disturbing influence induces by itself the calling forth of compensatory activity to neutralize or repair the disturbance. The higher in the scale of living beings, the more numerous, the more perfect and the more complicated do these regulatory agencies become. They tend to free the organism completely from the unfavorable influences and changes occurring in the environment.”

Léon Fredericq (1885) Cells and organisms strive for equilibrium or homeostasis, the maintenance of the steady states within acceptable ranges of physiological variables1,2_{. Achieving and sustaining homeostasis presents a constant}

endeavor as organisms face perpetual challenges, including but not limited to securing energy or food sources, maintaining hydration and osmotic stability or achieving optimal temperature. A perturbation or deviation from optimal conditions, commonly referred to as stress, presents as unfavorable. Therefore, a plethora of mechanisms exist at organismal and cellular level to sense, respond and repair the perturbation and regain homeostasis3_.

Mammalian cells, such as human or murine cells, can face a multitude of stresses with common examples such as genotoxic stress, oxidative stress, heat or cold shock or metabolic stresses (Figure 1). Once a cell senses a perturbation, various intricate mechanisms are set in motion to respond to the stress, neutralize it and repair any damage the stress may have caused. Typical stress responses involve intracellular DNA or proteins, or engagement of redox enzymes and redox scavengers4_{. Cellular signaling}

molecules, for example inflammatory proteins, or the adaptation of cell surface proteins with which a cell communicates to surrounding tissue cells or immune cells4_.

The cell cycle and DNA damage

One of the most dangerous and threatening stresses poses genotoxic stress leading to DNA damage. Common genotoxic agents include a multitude of chemicals, radiation or DNA-integrating viruses, however, most importantly, cell division manifests as major risk for DNA damage or DNA mutations at multiple stages of the cell cycle. DNA mutations (base mismatches, insertions or deletions) or DNA breaks commonly occur during DNA replication (S phase), while chromosome segregation errors during mitosis can result in DNA breaks or structural or numerical aneuploidy, the gain or loss of chromosomes5_{. Mutated or aneuploid cells}

Chapter 1

10

In order to ensure proper cell division, DNA replication and mitotic fidelity, the cell cycle is highly regulated and surveilled (Figure 2) alongside a plethora of safeguard mechanisms and DNA damage sensing and repair pathways. Central to cell cycle control are cyclin-dependent kinases (CDKs) and their activating

STRESS

Sense & respond

» Intracellular adaptation » Extracellular signaling

Stress relief

Stress management

AGING & DISEASES Homeostasis » Organismal homeostasis » Cellular homeostasis

–

+

–

+

Examples of stresses and perturbations:

• Genotoxic stress

• Mitotic stress & chromosome segregation errors • Oxidative stress

• Nutrient deficiency • Temperature sensitivity

Examples of stress responses:

• Cell cycle arrest

• Activation of cell cycle checkpoints • DNA damage repair response

• Engagement of redox proteins & scavenger molecules • Engagement of heat shock proteins or chaperons • Autophagy

• Secretion of signaling & inflammatory molecules • Adaptation of cell surface recognition proteins

Examples of “stress management” adaptations:

• Regulated cell death • Immunosurveillance

• Immune cell-mediated cell elimination • Durable proliferative arrest

Examples:

• Progeria

• Inflammation & inflammatory diseases • Metabolic diseases

• Neuro-degenerating diseases • Tumorigenesis

Figure 1: Maintenance of cellular and organismal homeostasis in response to stress. Deviations from optimal conditions,

stresses, are detrimental for cellular and organismal integrity. Consequently, cells and organisms developed a plethora of mechanisms to sense and respond to perturbations, and repair any damage the stress may have caused. Temporary stresses usually can be successfully relieved and repaired which re-establishes homeostasis. Irreparable or persistent stresses require additional adaptations to manage stress conditions. Typically, cells face two cell fates, cell death or cellular senescence. Despite that both outcomes implicate detrimental consequences for the cell itself, these cell fates benefit organismal homeostasis in the short-run. Failure to undergo either cell fate renders stressed, damaged cells prone to neoplastic transformation. Over extended periods, however, excessive cell death, cellular senescence and/or cell transformation can have serious deleterious consequences for the organism, as they drive aging, progeria, tumorigenesis and various other diseases.

factors, cyclins (e.g. Cyclin D1/D2 or Cyclin E1/E2), or inhibiting proteins, CDK inhibitors (e.g. P21/CDKN1A or P16/CDKN2A)6_{. This group of kinases phosphorylate various targets, most importantly the key regulators}

Retinoblastoma (RB) and family members Retinoblastoma-like 1 (RBL1/P107) and Retinoblastoma-like 2 (RBL2/P130), which upon phosphorylation are unable to inhibit E2F transcription factors and their downstream activation of target genes, which promote and support cell cycle progression6_.

Safeguard mechanisms, or cell cycle checkpoints assess cellular conditions and complications and pause cell cycle progression until conditions are optimal. The restriction point during G1 phase checks for

adequate cell size, nutrient and growth factor signaling and pre-existing DNA damage, while the G2/M

checkpoint examines for incomplete DNA replication and DNA damage7_{. The spindle assembly checkpoint}

during mitosis detects if chromosomes are properly aligned at the metaphase plate and attached at both kinetochores to microtubules emanating from the spindle poles7_{. Key players of the spindle assembly}

checkpoint include mitotic checkpoint complex members CDC20, MAD2, BUB3 and BUBR1.

Cell death or cellular senescence

If a stress is transient and repairable, cells can be highly proficient in adapting and overcoming the perturbation and returning to a homeostatic state (Figure 1). However, when facing chronic or irreparable stresses, cells are confronted to further adapt in the presence of the stress and often face two cellular fates: cell death or cellular senescence4_.

Spindle assembly checkpoint Restriction point G2/M checkpoint

G

M

S

G

G

Figure 2: The mammalian cell cycle. (Left) Phases of the cell cycle are tightly controlled by numerous proteins and signaling

pathways. Retinoblastoma protein (RB) and family members are key cell cycle progression regulators whose activity is controlled by cyclin dependent kinase (CDK)-cyclin complexes. Cell cycle checkpoints (red) are implemented as quality control steps to ensure completion of all prerequisites before a cell is allowed to enter the next phase of the cell cycle. (Right) CDK-cyclin complexes are in turn subject to regulation, predominantly inhibition, by CDK inhibitors of the INK4 family or CIP/KIP family with prominent members P16INK4A _{or P21}CIP1/WAF1_{, respectively.}

Cell death neutralizes a stressed cell permanently by intracellular or extracellular mechanisms or both. Intracellular mechanisms can include P53 activation and expression of pro-apoptotic genes, caspase activation, cytochrome c release, or DNA fragmentation, while extracellular events often include ligand-receptor interactions as part of extrinsic cell death pathways and immunosurveillance or immune-mediated cell killing mechanisms8_.

On the other hand, entry into cellular senescence permits a stressed cell to stay viable and metabolically active, while prohibiting cell division. This cellular state can be advantageous to limit excessive cellular loss after large scale stresses, or promote physiological reactions such as tissue remodeling and regeneration9-11_{, wound healing}12 _{or restrict excessive fibrosis}13-15_{. Induction of cellular}

senescence entails the concerted and prolonged activation of multiple CDK inhibitors (P21/CDKN1A and P16/CDKN2A) that establish a durable cell cycle arrest, as well as metabolic reprogramming and chromatin remodeling (Figure 3)16_{. Senescent cells often also exhibit activation of numerous signaling and secretory}

pathways that establish an intricate, bioactive secretory profile, the senescence-associated secretory phenotype (SASP), with which senescent cells interact with the microenvironment including the extracellular matrix, neighboring tissue cells and immune cells17_.

While cell death and cellular senescence can pose adverse outcomes for the cell itself, these mechanisms generally serve to sustain organismal homeostasis (Figure 1). Failure to undergo either cell fate can have detrimental consequences for the organism as these cells can be at risk for cellular transformation and subsequently tumor development.

Cell cycle arrest &

CDK inhibitor induction (P16, P21) Chromatin remodeling, DNA damage Lysosomal activity, SA-β-Gal activity Mitochondrial oxidative metabolism & ROS, Adaptations in BCL2 family members

Cytoplasmic chromatin fragments LaminB1 loss, HMGB1 relocalization

Senescence-associated secretory phenotype (SASP)

Figure 3: Cellular senescence represents a multifaceted cell fate with intricate cellular features. Senescent cells are typically

identified by a combination of characteristics, of which the most common ones are depicted. However, the phenotype of senescent cell subpopulations often presents as heterogenous, cell type- and context-dependent. CDK inhibitor, cyclin dependent kinase inhibitor; SA-β-Gal activity, senescence-associated-β-galactosidase activity; ROS, reactive oxygen species.

Chapter 1

Aging and age-related diseases

Paradoxically, chronic accumulation of senescent cells can also contribute to or cause unfavorable consequences for the organism, which include aging, progeria and numerous age-related diseases18,19_{. As}

such, cellular senescence is an example of antagonistic pleiotropy, in which certain traits enhance fitness early in life, therefore being favored by natural selection, but these same traits elicit unfavorable consequences in post-reproductive years when natural selection diminishes20_.

Over the past two decades, accumulation of senescent cells has been demonstrated in multiple human and murine tissues and organs and has been correlatively or causally linked to inflammation21,22_,

dysfunction and degeneration of tissue23_{, and fibrosis}24_{. Consequently, cellular senescence has been}

described to drive various diseases including but not limited to atherosclerosis25_{, osteoarthritis}26_,

Alzheimer’s Diseases27,28_{, Parkinson’s disease}29_{, progressive multiple sclerosis}30_{, Type 1 Diabetes}31_{, Type}

2 Diabetes32 _{and other metabolic or liver diseases}33,34_{. Senescent cells can also drive tumor initiation,}

progression and metastasis18,35-37_.

Chromosomal instability and cancer

Stress repair and relief, or stress management are critical to prevent propagation of damaged or mutated cells and therefore to counteract neoplastic transformation, tumor development and cancer. Certain stresses, however, can outcompete stress repair and management mechanisms. For example, perturbation of BUBR1 or other mitotic control and checkpoint genes has been shown to cause various mitotic defects including centrosome amplification and separation errors, mitotic spindle defects, chromosome missegregation, aneuploidy, often accompanied by chromosomal instability and eventually tumor development38-43_.

Aim and scope of this thesis

As our cells and organs are constantly subjected to a multitude of stresses most of which have life-threatening aspects, evolution has selected for sophisticated stress coping mechanisms. While these mechanisms are proficient in stress relief and repair, they are not infallible resulting in morbidities and mortality. Basic and biomedical research have made significant advances in describing stress response genes and pathways on molecular and physiological level, while being also successful in exploiting this knowledge for therapeutic purposes to combat pathologies and diseases. However, due to the variety and complexity of stresses and stress responses, it is still incompletely understood how the fate of cells, cell types, organs and ultimately organisms are shaped and determined by specific stresses. In particular, additional research is needed to understand the multifaceted interconnection of stress and stress responses during cellular senescence & aging and aneuploidy & tumorigenesis.

Chapter 2 provides an overview of the basic characteristics of cellular senescence, as well as

beneficial and detrimental properties of senescent cells with focus on kidney cells and pathologies. We also discuss how targeting of senescent cells, senotherapy, in the kidney may be leveraged to combat kidney diseases or kidney transplantation.

Chapter 1

14

In chapter 3 we review the complex relationship between cellular senescence and cancer. We

discuss that induction of cellular senescence in damaged, potentially pre-neoplastic cells is a potent anti-cancer mechanism, while chronically present senescent cells can promote all stages of tumor development and progression.

Chapter 4 focuses on the physiological aspects of detrimental senescent cells during metabolic

syndrome and metabolic diseases. We discuss how senescent cells can induce adipose tissue dysfunction and various liver pathologies.

In chapter 5 we investigate the stress-response gene, CDK inhibitor and cellular senescence key

player P21 (Cdkn1a) in vitro and mouse tissues. We describe a novel function of P21 and downstream target RB as mediators between the stress response aspects, cell cycle arrest and the cellular secretome that communicates with the microenvironment. We find that high levels of P21 are both required and sufficient to not only halt cell cycle progression and maintenance of cellular senescence aspects, but also induce immunosurveillance, particularly macrophage attraction. In the murine liver, this concerted effort allows for a spatiotemporal elimination of P21-induced hepatocytes.

In chapter 6 we assess the complex nature of BUBR1 functions in mosaic variegated aneuploidy

(MVA) progeria syndrome in mouse models. We find that the type of Bub1b allelic mutation and protein levels determine the type and severity of cellular phenotypes including cellular senescence, mitotic defects, chromosome segregation errors, as well as the type and severity of physiological consequences including progeria, aneuploidy and tumor development.

Chapter 7 describes our investigation of cyclin E1 (CCNE1) as a liver-selective oncogene that

promotes hepatocellular carcinoma development in mice. Our in vitro studies indicate that CCNE1 overexpression alters cell cycle timing, and causes DNA damage, chromosome missegregation and aneuploidy. In CCNE1-overexpressing mice, we discover that liver cells also suffer from chromosome segregation errors, near polyploid aneuploidy, chromosomal instability and are prone to tumor formation. We find that unlike in kidney or lung, CCNE1 overexpression in the liver provokes metabolic adaptations, oxidative stress, P53 activation and modulation of the immune system. While this study was performed in transgenic mice, our results may be clinically relevant as the CCNE1 gene is activated in hepatocellular carcinomas from patients infected with hepatitis B virus or adeno-associated virus type 2, due to integration of the virus near the CCNE1 gene.

In chapter 8 we demonstrate that Foxm1 acts as a haplo-insufficient tumor suppressor gene to

ensure mitotic fidelity, spindle symmetry and chromosome segregation in cells and mouse tissues. By using Foxm1 mutant cells and mice, we find that FOXM1 insufficiency results in hyperactivation of ECT2 and RhoA signaling leading to hyper-nucleation of cortical actin during mitosis and consequently asymmetric spindles that are prone to chromosome segregation errors. In mice, we find that FOXM1 insufficiency and RhoA-mediated cortical actin hyperactivation drive tumor development.

Finally, in chapter 9 we critically set our findings into context of the existing literature and discuss

References

1 Cannon, W. B. Organization for physiological homeostasis. Physiol Rev 9, 399–431 (1929).

2 Fredericq, L. Influence du milieu ambiant sur la composition du sang des animaux aquatiques. Arch. zool. exp. gen. III,

XXXIV-XXXVIII (1885).

3 Goldstein, D. S. & Kopin, I. J. Evolution of concepts of stress. Stress 10, 109-120, doi:10.1080/10253890701288935 (2007).

4 Galluzzi, L., Yamazaki, T. & Kroemer, G. Linking cellular stress responses to systemic homeostasis. Nat Rev Mol Cell Biol

19, 731-745, doi:10.1038/s41580-018-0068-0 (2018).

5 Hustedt, N. & Durocher, D. The control of DNA repair by the cell cycle. Nature cell biology 19, 1-9, doi:10.1038/ncb3452

(2016).

6 Asghar, U., Witkiewicz, A. K., Turner, N. C. & Knudsen, E. S. The history and future of targeting cyclin-dependent kinases in cancer therapy. Nat Rev Drug Discov 14, 130-146, doi:10.1038/nrd4504 (2015).

7 Barnum, K. J. & O'Connell, M. J. Cell cycle regulation by checkpoints. Methods Mol Biol 1170, 29-40,

doi:10.1007/978-1-4939-0888-2_2 (2014).

8 Galluzzi, L., Bravo-San Pedro, J. M., Kepp, O. & Kroemer, G. Regulated cell death and adaptive stress responses. Cell Mol

Life Sci 73, 2405-2410, doi:10.1007/s00018-016-2209-y (2016).

9 Saito, Y., Chikenji, T. S., Matsumura, T., Nakano, M. & Fujimiya, M. Exercise enhances skeletal muscle regeneration by promoting senescence in fibro-adipogenic progenitors. Nat Commun 11, 889, doi:10.1038/s41467-020-14734-x (2020).

10 Brighton, P. J. et al. Clearance of senescent decidual cells by uterine natural killer cells in cycling human endometrium. Elife

6, doi:10.7554/eLife.31274 (2017).

11 Munoz-Espin, D. et al. Programmed cell senescence during mammalian embryonic development. Cell 155, 1104-1118,

doi:10.1016/j.cell.2013.10.019 (2013).

12 Demaria, M. et al. An essential role for senescent cells in optimal wound healing through secretion of PDGF-AA. Dev Cell

31, 722-733, doi:10.1016/j.devcel.2014.11.012 (2014).

13 Krizhanovsky, V. et al. Senescence of activated stellate cells limits liver fibrosis. Cell 134, 657-667,

doi:10.1016/j.cell.2008.06.049 (2008).

14 Zhu, F. et al. Senescent cardiac fibroblast is critical for cardiac fibrosis after myocardial infarction. PloS one 8, e74535,

doi:10.1371/journal.pone.0074535 (2013).

15 Jun, J. I. & Lau, L. F. The matricellular protein CCN1 induces fibroblast senescence and restricts fibrosis in cutaneous wound healing. Nature cell biology 12, 676-685, doi:10.1038/ncb2070 (2010).

16 Gorgoulis, V. et al. Cellular Senescence: Defining a Path Forward. Cell 179, 813-827, doi:10.1016/j.cell.2019.10.005 (2019).

17 Faget, D. V., Ren, Q. & Stewart, S. A. Unmasking senescence: context-dependent effects of SASP in cancer. Nat Rev

Cancer 19, 439-453, doi:10.1038/s41568-019-0156-2 (2019).

18 Baker, D. J. et al. Naturally occurring p16(Ink4a)-positive cells shorten healthy lifespan. Nature 530, 184-189,

doi:10.1038/nature16932 (2016).

19 Baker, D. J. et al. Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders. Nature 479,

232-236, doi:10.1038/nature10600 (2011).

20 Giaimo, S. & d'Adda di Fagagna, F. Is cellular senescence an example of antagonistic pleiotropy? Aging Cell 11, 378-383,

doi:10.1111/j.1474-9726.2012.00807.x (2012).

21 Dou, Z. et al. Cytoplasmic chromatin triggers inflammation in senescence and cancer. Nature 550, 402-406,

doi:10.1038/nature24050 (2017).

22 Pribluda, A. et al. A senescence-inflammatory switch from cancer-inhibitory to cancer-promoting mechanism. Cancer cell

24, 242-256, doi:10.1016/j.ccr.2013.06.005 (2013).

23 Ritschka, B. et al. The senotherapeutic drug ABT-737 disrupts aberrant p21 expression to restore liver regeneration in adult mice. Genes & development 34, 489-494, doi:10.1101/gad.332643.119 (2020).

24 Anderson, R. et al. Length-independent telomere damage drives post-mitotic cardiomyocyte senescence. EMBO J 38,

doi:10.15252/embj.2018100492 (2019).

25 Childs, B. G. et al. Senescent intimal foam cells are deleterious at all stages of atherosclerosis. Science 354, 472-477,

doi:10.1126/science.aaf6659 (2016).

26 Jeon, O. H. et al. Local clearance of senescent cells attenuates the development of post-traumatic osteoarthritis and creates a pro-regenerative environment. Nature medicine 23, 775-781, doi:10.1038/nm.4324 (2017).

27 Bussian, T. J. et al. Clearance of senescent glial cells prevents tau-dependent pathology and cognitive decline. Nature 562,

578-582, doi:10.1038/s41586-018-0543-y (2018).

28 Zhang, P. et al. Senolytic therapy alleviates Abeta-associated oligodendrocyte progenitor cell senescence and cognitive deficits in an Alzheimer's disease model. Nat Neurosci 22, 719-728, doi:10.1038/s41593-019-0372-9 (2019).

29 Chinta, S. J. et al. Cellular Senescence Is Induced by the Environmental Neurotoxin Paraquat and Contributes to Neuropathology Linked to Parkinson's Disease. Cell reports 22, 930-940, doi:10.1016/j.celrep.2017.12.092 (2018).

30 Nicaise, A. M. et al. Cellular senescence in progenitor cells contributes to diminished remyelination potential in progressive multiple sclerosis. Proceedings of the National Academy of Sciences of the United States of America 116, 9030-9039,

doi:10.1073/pnas.1818348116 (2019).

31 Thompson, P. J. et al. Targeted Elimination of Senescent Beta Cells Prevents Type 1 Diabetes. Cell Metab 29, 1045-1060

Chapter 1

16

32 Aguayo-Mazzucato, C. et al. Acceleration of beta Cell Aging Determines Diabetes and Senolysis Improves Disease Outcomes. Cell Metab 30, 129-142 e124, doi:10.1016/j.cmet.2019.05.006 (2019).

33 Ogrodnik, M. et al. Obesity-Induced Cellular Senescence Drives Anxiety and Impairs Neurogenesis. Cell Metab, doi:10.1016/j.cmet.2018.12.008 (2019).

34 Ogrodnik, M. et al. Cellular senescence drives age-dependent hepatic steatosis. Nat Commun 8, 15691,

doi:10.1038/ncomms15691 (2017).

35 Toso, A. et al. Enhancing chemotherapy efficacy in Pten-deficient prostate tumors by activating the senescence-associated antitumor immunity. Cell reports 9, 75-89, doi:10.1016/j.celrep.2014.08.044 (2014).

36 Krtolica, A., Parrinello, S., Lockett, S., Desprez, P. Y. & Campisi, J. Senescent fibroblasts promote epithelial cell growth and tumorigenesis: a link between cancer and aging. Proceedings of the National Academy of Sciences of the United States of

America 98, 12072-12077, doi:10.1073/pnas.211053698 (2001).

37 Demaria, M. et al. Cellular Senescence Promotes Adverse Effects of Chemotherapy and Cancer Relapse. Cancer Discov

7, 165-176, doi:10.1158/2159-8290.CD-16-0241 (2017).

38 Jeganathan, K., Malureanu, L., Baker, D. J., Abraham, S. C. & van Deursen, J. M. Bub1 mediates cell death in response to

chromosome missegregation and acts to suppress spontaneous tumorigenesis. J Cell Biol 179, 255-267,

doi:10.1083/jcb.200706015 (2007).

39 Nam, H. J. & van Deursen, J. M. Cyclin B2 and p53 control proper timing of centrosome separation. Nature cell biology 16,

538-549, doi:10.1038/ncb2952 (2014).

40 Weaver, R. L. et al. BubR1 alterations that reinforce mitotic surveillance act against aneuploidy and cancer. Elife 5,

doi:10.7554/eLife.16620 (2016).

41 Zhang, Y. et al. USP44 regulates centrosome positioning to prevent aneuploidy and suppress tumorigenesis. The Journal

of clinical investigation 122, 4362-4374, doi:10.1172/JCI63084 (2012).

42 Baker, D. J. et al. BubR1 insufficiency causes early onset of aging-associated phenotypes and infertility in mice. Nature

genetics 36, 744-749, doi:10.1038/ng1382 (2004).

43 Hanks, S. et al. Constitutional aneuploidy and cancer predisposition caused by biallelic mutations in BUB1B. Nature genetics