Therapy-induced polyploidization and senescence: Coincidence or interconnection?

Polyploid somatic cells have 'programmed' roles in normal development and stress responses. Transient polyploidy states have been observed in several tumor types at early stages of tumorigenesis. They can give rise to the aneuploidy state which is a common feature of human cancer cells. Similarly, to cancer development, cancer treatment can lead to transient polyploidy. Polyploid giant cells (PGCCs) in cancer are often associated with poor prognosis and disease relapse. Cancer cell senescence- a proliferation arrest accompanied by a set of characteristic markers- induced by therapy is also associated with transient polyploidy formation and cancer relapse. The question is whether therapy-induced senescence (TIS) and therapy induced polyploidy (TIP) are mechanistically or coincidentally connected. This problem needs to be solved rather urgently, because TIS appears to be more common phenomena than originally believed. Another arising question concerns reversibility of cancer cell senescence as a consequence of atypical divisions of polyploid cells. In our review we will try to answer this fundamental question by referring to published literature and to our own studies.

Improved Autophagic Flux in Escapers from Doxorubicin-Induced Senescence/Polyploidy of Breast Cancer Cells.

The induction of senescence/polyploidization and their role in cancer recurrence is still a poorly explored issue. We showed that MDA-MB-231 and MCF-7 breast cancer cells underwent reversible senescence/polyploidization upon pulse treatment with doxorubicin (dox). Subsequently, senescent/polyploid cells produced progeny (escapers) that possessed the same amount of DNA as parental cells. In a dox-induced senescence/polyploidization state, the accumulation of autophagy protein markers, such as LC3B II and p62/SQSTM1, was observed. However, the senescent cells were characterized by a very low rate of new autophagosome formation and degradation, estimated by autophagic index. In contrast to senescent cells, escapers had a substantially increased autophagic index and transcription factor EB activation, but a decreased level of an autophagy inhibitor, Rubicon, and autophagic vesicles with non-degraded cargo. These results strongly suggested that autophagy in escapers was improved, especially in MDA-MB-231 cells. The escapers of both cell lines were also susceptible to dox-induced senescence. However, MDA-MB-231 cells which escaped from senescence were characterized by a lower number of γH2AX foci and a different pattern of interleukin synthesis than senescent cells. Thus, our studies showed that breast cancer cells can undergo senescence uncoupled from autophagy status, but autophagic flux resumption may be indispensable in cancer cell escape from senescence/polyploidy.

[Neuronal ageing]. / Starzenie neuronów.

Ageing leads to irreversible alterations in the nervous system, which to various extent impair its functions such as capacity to learn and memory. In old neurons and brain, similarly to what may take place in other cells, there is increased oxidative stress, disturbed energetic homeostasis and metabolism, accumulation of damage in proteins and nucleic acids. Characteristic of old neurons are alterations in plasticity, synaptic transmission, sensitivity to neurotrophic factors and cytoskeletal changes. Some markers of senescence, whose one of them is SA-beta-galactosidase were used to show the process of neuronal ageing both in vitro, and in vivo. Some research suggest that, despite the fact that neurons are postmitotic cells, it is cell cycle proteins which play a certain role in their biology, e.g. differentiation. However, their role in neuronal ageing is not known or explained. Ageing is the serious factor of development of neurodegenerative diseases among others Alzheimer disease.

Unbiased proteomic analysis of extracellular vesicles secreted by senescent human vascular smooth muscle cells reveals their ability to modulate immune cell functions.

Atherosclerosis, a common age-related disease, is characterized by intense immunological activity. Atherosclerotic plaque is composed of endothelial cells, vascular smooth muscle cells (VSMCs), lipids and immune cells infiltrating from the blood. During progression of the disease, VSMCs undergo senescence within the plaque and secrete SASP (senescence-associated secretory phenotype) factors that can actively modulate plaque microenvironment. We demonstrated that senescent VSMCs secrete increased number of extracellular vesicles (senEVs). Based on unbiased proteomic analysis of VMSC-derived EVs and of the soluble fraction of SASP (sSASP), more than 900 proteins were identified in each of SASP compartments. Comparison of the composition of VMSC-derived EVs with the SASP atlas revealed several proteins, including Serpin Family F Member 1 (SERPINF1) and Thrombospondin 1 (THBS1), as commonly upregulated components of EVs secreted by senescent VSMCs and fibroblasts. Among soluble SASP factors, only Growth Differentiation Factor 15 (GDF15) was universally increased in the secretome of senescent VSMCs, fibroblasts, and epithelial cells. Bioinformatics analysis of EV proteins distinguished functionally organized protein networks involved in immune cell function regulation. Accordingly, EVs released by senescent VSMCs induced secretion of IL-17, INFγ, and IL-10 by T cells and of TNFα produced by monocytes. Moreover senEVs influenced differentiation of monocytes favoring mix M1/M2 polarization with proinflammatory characteristics. Altogether, our studies provide a complex, unbiased analysis of VSMC SASP and prove that EVs derived from senescent VSMCs influence the cytokine milieu by modulating immune cell activity. Our results strengthen the role of senescent cells as an important inducer of inflammation in atherosclerosis.

ATM-deficient neural precursors develop senescence phenotype with disturbances in autophagy.

ATM is a kinase involved in DNA damage response (DDR), regulation of response to oxidative stress, autophagy and mitophagy. Mutations in the ATM gene in humans result in ataxi A-Telangiectasia disease (A-T) characterized by a variety of symptoms with neurodegeneration and premature ageing among them. Since brain is one of the most affected organs in A-T, we have focused on senescence of neural progenitor cells (NPCs) derived from A-T reprogrammed fibroblasts. Accordingly, A-T NPCs obtained through neural differentiation of iPSCs in 5% oxygen possessed some features of senescence including increased activity of SA-ß-gal and secretion of IL6 and IL8 in comparison to control NPCs. This phenotype of A-T NPC was accompanied by elevated oxidative stress. A-T NPCs exhibited symptoms of impaired autophagy and mitophagy with lack of response to chloroquine treatment. Additional sources of oxidative stress like increased oxygen concentration (20 %) and H2O2 respectively aggravated the phenotype of senescence and additionally disturbed the process of mitophagy. In both cases only A-T NPCs reacted to the treatment. We conclude that oxidative stress may be responsible for the phenotype of senescence and impairment of autophagy in A-T NPCs. Our results point to senescent A-T cells as a potential therapeutic target in this disease.

NOX4 downregulation leads to senescence of human vascular smooth muscle cells.

Senescence is a stress response characterized by an irreversible growth arrest and alterations in certain cell functions. It is believed that both double-strand DNA breaks (DSB) and increased ROS level are the main culprit of senescence. Excessive ROS production is also particularly important in the development of a number of cardiovascular disorders. In this context the involvement of professional ROS-producing enzymes, NADPH oxidases (NOX), was postulated. In contrary to the common knowledge, we have shown that not only increased ROS production but also diminished ROS level could be involved in the induction of senescence.Accordingly, our studies revealed that stress-induced premature senescence (SIPS) of vascular smooth muscle cells (VSMCs) induced by doxorubicin or H2O2, correlates with increased level of DSB and ROS. On the other hand, both SIPS and replicative senescence were accompanied by diminished expression of NOX4. Moreover, inhibition of NOX activity or decrease of NOX4 expression led to permanent growth arrest of VSMCs and secretion of interleukins and VEGF. Interestingly, cells undergoing senescence due to NOX4 depletion neither acquired DSB nor activated DNA damage response. Instead, transient induction of the p27, upregulation of HIF-1alpha, decreased expression of cyclin D1 and hypophosphorylated Rb was observed. Our results showed that lowering the level of ROS-producing enzyme - NOX4 oxidase below physiological level leads to cellular senescence of VSMCs which is correlated with secretion of pro-inflammatory cytokines. Thus the use of specific NOX4 inhibitors for pharmacotherapy of vascular diseases should be carefully considered.

Is senescence-associated ß-galactosidase a marker of neuronal senescence?

One of the features of cellular senescence is the activity of senescence-associated- ß-galactosidase (SA-ß-gal). The main purpose of this study was to evaluate this marker of senescence in aging neurons. We found that cortical neurons exhibited noticeable SA-ß-gal activity quite early in culture. Many SA-ß-gal-positive neurons were negative for another canonical marker of senescence, namely, double-strand DNA breaks (DSBs). Moreover, DDR signalling triggered by low doses of doxorubicin did not accelerate the appearance of neuronal SA-ß-gal. In vivo, we observed pronounced induction of SA-ß-gal activity in the hippocampus of 24-month-old mice, which is consistent with previous findings and supports the view that at this advanced age neurons developed a senescence-like phenotype. Surprisingly however, relatively high SA-ß-gal activity, probably unrelated to the senescence process, was also observed in much younger, 3-month-old mice. In conclusion, we propose that SA-ß-gal activity in neurons cannot be attributed uniquely to cell senescence either in vitro or in vivo. Additionally, we showed induction of REST protein in aging neurons in long-term culture and we propose that REST could be a marker of neuronal senescence in vitro.

Curcumin-treated cancer cells show mitotic disturbances leading to growth arrest and induction of senescence phenotype.

Cellular senescence is recognized as a potent anticancer mechanism that inhibits carcinogenesis. Cancer cells can also undergo senescence upon chemo- or radiotherapy. Curcumin, a natural polyphenol derived from the rhizome of Curcuma longa, shows anticancer properties both in vitro and in vivo. Previously, we have shown that treatment with curcumin leads to senescence of human cancer cells. Now we identified the molecular mechanism underlying this phenomenon. We observed a time-dependent accumulation of mitotic cells upon curcumin treatment. The time-lapse analysis proved that those cells progressed through mitosis for a significantly longer period of time. A fraction of cells managed to divide or undergo mitotic slippage and then enter the next phase of the cell cycle. Cells arrested in mitosis had an improperly formed mitotic spindle and were positive for γH2AX, which shows that they acquired DNA damage during prolonged mitosis. Moreover, the DNA damage response pathway was activated upon curcumin treatment and the components of this pathway remained upregulated while cells were undergoing senescence. Inhibition of the DNA damage response decreased the number of senescent cells. Thus, our studies revealed that the induction of cell senescence upon curcumin treatment resulted from aberrant progression through the cell cycle. Moreover, the DNA damage acquired by cancer cells, due to mitotic disturbances, activates an important molecular mechanism that determines the potential anticancer activity of curcumin.

Polyploidy Formation in Doxorubicin-Treated Cancer Cells Can Favor Escape from Senescence.

Cancer cells can undergo stress-induced premature senescence, which is considered to be a desirable outcome of anticancer treatment. However, the escape from senescence and cancer cell repopulation give rise to some doubts concerning the effectiveness of the senescence-induced anticancer therapy. Similarly, it is postulated that polyploidization of cancer cells is connected with disease relapse. We postulate that cancer cell polyploidization associated with senescence is the culprit of atypical cell divisions leading to cancer cell regrowth. Accordingly, we aimed to dissociate between these two phenomena. We induced senescence in HCT 116 cells by pulse treatment with doxorubicin and observed transiently increased ploidy, abnormal nuclear morphology, and various distributions of some proteins (e.g., p21, Ki-67, SA-ß-galactosidase) in the subnuclei. Doxorubicin-treated HCT 116 cells displayed an increased production of reactive oxygen species (ROS) possibly caused by an increased amount of mitochondria, which are characterized by low membrane potential. A decrease in the level of ROS by Trolox partially protected the cells from polyploidization but not from senescence. Interestingly, a decreased level of ROS prevented the cells from escaping senescence. We also show that MCF7 cells senesce, but this is not accompanied by the increase of ploidy upon doxorubicin treatment. Moreover, they were stably growth arrested, thus proving that polyploidy but not senescence per se enables to regain the ability to proliferate. Our preliminary results indicate that the different propensity of the HCT 116 and MCF7 cells to increase ploidy upon cell senescence could be caused by a different level of the mTOR and/or Pim-1 kinases.