Tumor-Associated Senescent Macrophages, Their Markers, and Their Role in Tumor Microenvironment.

Tumor-associated macrophages (TAMs) are an important component of the tumor microenvironment (TME) and the most abundant population of immune cells infiltrating a tumor. TAMs can largely determine direction of anti-tumor immune response by promoting it or, conversely, contribute to formation of an immunosuppressive TME that allows tumors to evade immune control. Through interactions with tumor cells or other cells in the microenvironment and, as a result of action of anti-cancer therapy, macrophages can enter senescence. In this review, we have attempted to summarize information available in the literature on the role of senescent macrophages in tumors. With the recent development of senolytic therapeutic strategies aimed at removing senescent cells from an organism, it seems important to discuss functions of the senescent macrophages and potential role of the senolytic drugs in reprogramming TAMs to enhance anti-tumor immune response and improve efficacy of cancer treatment.

The Differential Effect of Senolytics on SASP Cytokine Secretion and Regulation of EMT by CAFs.

The tumor microenvironment (TME) plays an essential role in tumor progression and in modulating tumor response to anticancer therapy. Cellular senescence leads to a switch in the cell secretome, characterized by the senescence-associated secretory phenotype (SASP), which may regulate tumorigenesis. Senolytic therapy is considered a novel anticancer strategy that eliminates the deleterious effects of senescent cells in the TME. Here, we show that two different types of senolytic drugs, despite efficiently depleting senescent cells, have opposite effects on cancer-associated fibroblasts (CAFs) and their ability to regulate epithelial-mesenchymal transition (EMT). We found that senolytic drugs, navitoclax and the combination of dasatinib/quercetin, reduced the number of spontaneously senescent and TNF-induced senescent CAFs. Despite the depletion of senescent cells, the combination of dasatinib/quercetin versus navitoclax increased the secretion of the SASP pro-inflammatory cytokine IL-6. This differential effect correlated with the promotion of enhanced migration and EMT in MC38 colorectal cancer cells. Our results demonstrate that some senolytics may have side effects unrelated to their senolytic activity and may promote tumorigenesis. We argue for more careful and extensive studies of the effects of senolytics on various aspects of tumor progression and tumor resistance to therapy before the senolytic strategy is implemented in the clinic.

Clonal haematopoiesis - a novel entity that modifies pathological processes in elderly.

Progress in the development of new sequencing techniques with wider accessibility and higher sensitivity of the protocol of deciphering genome particularities led to the discovery of a new phenomenon - clonal haematopoiesis. It is characterized by the presence in the bloodstream of elderly people a minor clonal population of cells with mutations in certain genes, but without any sign of disease related to the hematopoietic system. Here we will review this recent advancement in the field of clonal haematopoiesis and how it may affect the disease's development in old age.

p16High senescence restricts cellular plasticity during somatic cell reprogramming.

Despite advances in four-factor (4F)-induced reprogramming (4FR) in vitro and in vivo, how 4FR interconnects with senescence remains largely under investigated. Here, using genetic and chemical approaches to manipulate senescent cells, we show that removal of p16High cells resulted in the 4FR of somatic cells into totipotent-like stem cells. These cells expressed markers of both pluripotency and the two-cell embryonic state, readily formed implantation-competent blastoids and, following morula aggregation, contributed to embryonic and extraembryonic lineages. We identified senescence-dependent regulation of nicotinamide N-methyltransferase as a key mechanism controlling the S-adenosyl-L-methionine levels during 4FR that was required for expression of the two-cell genes and acquisition of an extraembryonic potential. Importantly, a partial 4F epigenetic reprogramming in old mice was able to reverse several markers of liver aging only in conjunction with the depletion of p16High cells. Our results show that the presence of p16High senescent cells limits cell plasticity, whereas their depletion can promote a totipotent-like state and histopathological tissue rejuvenation during 4F reprogramming.

RUNX1/CEBPA Mutation in Acute Myeloid Leukemia Promotes Hypermethylation and Indicates for Demethylation Therapy.

Acute myeloid leukemia (AML) is a rapidly progressing heterogeneous disease with a high mortality rate, which is characterized by hyperproliferation of atypical immature myeloid cells. The number of AML patients is expected to increase in the near future, due to the old-age-associated nature of AML and increased longevity in the human population. RUNX1 and CEBPA, key transcription factors (TFs) of hematopoiesis, are frequently and independently mutated in AML. RUNX1 and CEBPA can bind TET2 demethylase and attract it to their binding sites (TFBS) in cell lines, leading to DNA demethylation of the regions nearby. Since TET2 does not have a DNA-binding domain, TFs are crucial for its guidance to target genomic locations. In this paper, we show that RUNX1 and CEBPA mutations in AML patients affect the methylation of important regulatory sites that resulted in the silencing of several RUNX1 and CEBPA target genes, most likely in a TET2-dependent manner. We demonstrated that hypermethylation of TFBS in AML cells with RUNX1 mutations was associated with resistance to anticancer chemotherapy. Demethylation therapy restored expression of the RUNX1 target gene, BIK, and increased sensitivity of AML cells to chemotherapy. If our results are confirmed, mutations in RUNX1 could be an indication for prescribing the combination of cytotoxic and demethylation therapies.

Inhibition of the DNA damage response phosphatase PPM1D reprograms neutrophils to enhance anti-tumor immune responses.

PPM1D/Wip1 is a negative regulator of the tumor suppressor p53 and is overexpressed in several human solid tumors. Recent reports associate gain-of-function mutations of PPM1D in immune cells with worse outcomes for several human cancers. Here we show that mice with genetic knockout of Ppm1d or with conditional knockout of Ppm1d in the hematopoietic system, in myeloid cells, or in neutrophils all display significantly reduced growth of syngeneic melanoma or lung carcinoma tumors. Ppm1d knockout neutrophils infiltrate tumors extensively. Chemical inhibition of Wip1 in human or mouse neutrophils increases anti-tumor phenotypes, p53-dependent expression of co-stimulatory ligands, and proliferation of co-cultured cytotoxic T cells. These results suggest that inhibition of Wip1 in neutrophils enhances immune anti-tumor responses.

Selecting the first chemical molecule inhibitor of HSP110 for colorectal cancer therapy.

Pro-survival stress-inducible chaperone HSP110 is the only HSP for which a mutation has been found in a cancer. Multicenter clinical studies demonstrated a direct association between HSP110 inactivating mutation presence and excellent prognosis in colorectal cancer patients. Here, we have combined crystallographic studies on human HSP110 and in silico modeling to identify HSP110 inhibitors that could be used in colorectal cancer therapy. Two molecules (foldamers 33 and 52), binding to the same cleft of HSP110 nucleotide-binding domain, were selected from a chemical library (by co-immunoprecipitation, AlphaScreening, Interference-Biolayer, Duo-link). These molecules block HSP110 chaperone anti-aggregation activity and HSP110 association to its client protein STAT3, thereby inhibiting STAT3 phosphorylation and colorectal cancer cell growth. These effects were strongly decreased in HSP110 knockdown cells. Foldamer's 33 ability to inhibit tumor growth was confirmed in two colorectal cancer animal models. Although tumor cell death (apoptosis) was noted after treatment of the animals with foldamer 33, no apparent toxicity was observed, notably in epithelial cells from intestinal crypts. Taken together, we identified the first HSP110 inhibitor, a possible drug-candidate for colorectal cancer patients whose unfavorable outcome is associated to HSP110.

HSP110 translocates to the nucleus upon genotoxic chemotherapy and promotes DNA repair in colorectal cancer cells.

A multicenter clinical study demonstrated the presence of a loss-of-function HSP110 mutation in about 15% of colorectal cancers, which resulted from an alternative splicing and was produced at the detriment of wild-type HSP110. Patients expressing low levels of wild-type HSP110 had excellent outcomes (i.e. response to an oxaliplatin-based chemotherapy). Here, we show in vitro, in vivo, and in patients' biopsies that HSP110 co-localizes with DNA damage (γ-H2AX). In colorectal cancer cells, HSP110 translocates into the nucleus upon treatment with genotoxic chemotherapy such as oxaliplatin. Furthermore, we show that HSP110 interacts with the Ku70/Ku80 heterodimer, an essential element of the non-homologous end joining (NHEJ) repair machinery. We also demonstrate by evaluating the resolved 53BP1 foci that depletion in HSP110 impairs repair steps of the NHEJ pathway, which is associated with an increase in DNA double-strand breaks and in the cells' sensitivity to oxaliplatin. HSP110-depleted cells sensitization to oxaliplatin-induced DNA damage is abolished upon re-expression of HSP110. Confirming a role for HSP110 in DNA non-homologous repair, SCR7 and NU7026, two inhibitors of the NHEJ pathway, circumvents HSP110-induced resistance to chemotherapy. In conclusion, HSP110 through its interaction with the Ku70/80 heterodimer may participate in DNA repair, thereby inducing a protection against genotoxic therapy.

Wip1 phosphatase: between p53 and MAPK kinases pathways.

Cells undergoing oncogenic transformation frequently inactivate tumor suppressor pathways that could prevent their uncontrolled growth. Among those pathways p53 and p38MAPK pathways play a critical role in regulation of cell cycle, senescence and cell death in response to activation of oncogenes, stress and DNA damage. Consequently, these two pathways are important in determining the sensitivity of tumor cells to anti-cancer treatment. Wild type p53-induced phosphatase, Wip1, is involved in governance of both pathways. Recently, strategies directed to manipulation with Wip1 activity proposed to advance current day anticancer treatment and novel chemical compounds synthesized to improve specificity of manipulation with Wip1 activity. Here we reviewed the history of Wip1 studies in vitro and in vivo, in genetically modified animal models that support Wip1 role in tumorigenesis through regulation of p53 and p38MAPK pathways. Based on our knowledge we propose several recommendations for future more accurate studies of Wip1 interactions with other pathways involved in tumorigenesis using recently developed tools and for adoption of Wip1 manipulation strategies in anti-cancer therapy.

Phosphatase WIP1 regulates adult neurogenesis and WNT signaling during aging.

The number of newly formed neurons declines rapidly during aging, and this decrease in neurogenesis is associated with decreased function of neural stem/progenitor cells (NPCs). Here, we determined that a WIP1-dependent pathway regulates NPC differentiation and contributes to the age-associated decline of neurogenesis. Specifically, we found that WIP1 is expressed in NPCs of the mouse subventricular zone (SVZ) and aged animals with genetically enhanced WIP1 expression exhibited higher NPC numbers and neuronal differentiation compared with aged WT animals. Additionally, augmenting WIP1 expression in aged animals markedly improved neuron formation and rescued a functional defect in fine odor discrimination in aged mice. We identified the WNT signaling pathway inhibitor DKK3 as a key downstream target of WIP1 and found that expression of DKK3 in the SVZ is restricted to NPCs. Using murine reporter strains, we determined that DKK3 inhibits neuroblast formation by suppressing WNT signaling and Dkk3 deletion or pharmacological activation of the WNT pathway improved neuron formation and olfactory function in aged mice. We propose that WIP1 controls DKK3-dependent inhibition of neuronal differentiation during aging and suggest that regulating WIP1 levels could prevent certain aspects of functional decline of the aging brain.

Inhibition of HSP70: a challenging anti-cancer strategy.

HSP70 is a chaperone that accumulates in the cells after many different stresses promoting cell survival in response to the adverse conditions. In contrast to normal cells, most cancer cells abundantly express HSP70 at the basal level to resist to various insults at different stages of tumorigenesis and during anti-cancer treatment. This cancer cells addiction for HSP70 is the rational for its targeting in cancer therapy. Much effort has been dedicated in the last years for the active search of HSP70 inhibitors. Additionally, the recent clinical trials on highly promising inhibitors of another stress protein, HSP90, showed compensatory increase in HSP70 levels and raised the question of necessity to combine HSP90 inhibitors with simultaneous inhibition of HSP70. Here we analyzed the recent advancement in creation of novel HSP70 inhibitors and different strategies for their use in anti-cancer therapy.

Wip1 sensitizes p53-negative tumors to apoptosis by regulating the Bax/Bcl-xL ratio.

Wip1 is a stress-response phosphatase that negatively regulates several tumor suppressors, including p53. In a sizeable fraction of tumors, overexpression or amplification of Wip1 compromises p53 functions; inhibition of Wip1 activity is an attractive strategy for improving treatment of these tumors. However, over half of human tumors contain mutations in the p53 gene or have lost both alleles. Recently, we observed that in cancer cells lacking wild type p53, reduction of Wip1 expression was ineffective, whereas, surprisingly, overexpression of Wip1 increased anticancer drug sensitivity. The increased sensitivity resulted from activation of the intrinsic pathway of apoptosis through increased levels of the pro-apoptotic protein Bax and decreased levels of the anti-apoptotic protein Bcl-xL. We showed that interaction of Wip1 and the transcription factor RUNX2, specifically through dephosphorylation of RUNX2 phospho-S432, resulted in increased expression of Bax. Interestingly, overexpression of Wip1 increased drug sensitivity only in the p53-negative tumor cells while protecting the wild type p53-containing normal cells from drug-induced collateral injury. Here, we provide evidence that Wip1 overexpression decreases expression of Bcl-xL through negative regulation of NFκB activity. Thus, Wip1 overexpression increases the sensitivity of p53-negative cancer cells to anticancer drugs by separately affecting Bax and Bcl-xL protein levels.

Wip1 promotes RUNX2-dependent apoptosis in p53-negative tumors and protects normal tissues during treatment with anticancer agents.

The inactivation of the p53 tumor suppressor pathway in many cancers often increases their resistance to anticancer therapy. Here we show that a previously proposed strategy directed to Wip1 inhibition could be ineffective in tumors lacking p53. On the contrary, Wip1 overexpression sensitized these tumors to chemotherapeutic agents. This effect was mediated through interaction between Wip1 and RUNX2 that resulted, in response to anticancer treatment, in RUNX2-dependent transcriptional induction of the proapoptotic Bax protein. The potentiating effects of Wip1 overexpression on chemotherapeutic agents were directed only to tumor cells lacking p53. The overexpression of Wip1 in normal tissues provided protection from cisplatin-induced apoptosis through decreased strength of upstream signaling to p53. Thus, Wip1 phosphatase promotes apoptosis in p53-negative tumors and protects normal tissues during treatment with anticancer agents.

Peptides and aptamers targeting HSP70: a novel approach for anticancer chemotherapy.

The inhibition of heat shock protein 70 (HSP70) is an emerging strategy in cancer therapy. Unfortunately, no specific inhibitors are clinically available. By yeast two-hybrid screening, we have identified multiple peptide aptamers that bind HSP70. When expressed in human tumor cells, two among these peptide aptamers-A8 and A17-which bind to the peptide-binding and the ATP-binding domains of HSP70, respectively, specifically inhibited the chaperone activity, thereby increasing the cells' sensitivity to apoptosis induced by anticancer drugs. The 13-amino acid peptide from the variable region of A17 (called P17) retained the ability to specifically inhibit HSP70 and induced the regression of subcutaneous tumors in vivo after local or systemic injection. This antitumor effect was associated with an important recruitment of macrophages and T lymphocytes into the tumor bed. Altogether, these data indicate that peptide aptamers or peptides that target HSP70 may be considered as novel lead compounds for cancer therapy.

p38MAPK controls expression of multiple cell cycle inhibitors and islet proliferation with advancing age.

Aging is a complex organismal process that is controlled by genetic, environmental, and behavioral factors. Accumulating evidence supports a role for different cell cycle inhibitors in mammalian aging. Little is known, however, about the upstream signals that induce their expression. Here, we explore the role of p38MAPK by generating a dominant-negative allele (p38(AF)) in which activating phosphorylation sites Thr180 and Tyr182 are mutated. Heterozygous p38(AF) mice show a marked attenuation of p38-dependent signaling and age-induced expression of multiple cell cycle inhibitors in different organs, including pancreatic islets. As a result, aged p38(AF/+) mice show enhanced proliferation and regeneration of islets when compared to wild-type littermates. We further find an age-related reduction in expression of the p38-specific phosphatase Wip1. Wip1-deficient mice demonstrate decreased islet proliferation, while Wip1 overexpression rescues aging-related decline in proliferation and regenerative capacity. We propose that modulation of p38MAPK activity may provide new avenues for treating certain age-related degenerative diseases.

Wip1 regulates the generation of new neural cells in the adult olfactory bulb through p53-dependent cell cycle control.

Continual generation of new neural cells from adult neural stem/progenitor cells (NPCs) is an important component of life-long brain plasticity. However, the intrinsic regulation of this process remains poorly defined. Here we report that Wip1 phosphatase, previously studied in oncogenesis, functions as a crucial physiological regulator in adult neural cell generation. Wip1 deficiency resulted in a 90% decrease in new cell formation in adult olfactory bulb, accompanied by aberrantly decreased NPC amplification, stem cell frequency, and self-renewal. At a cellular level, Wip1 knockout NPCs exhibit a prolonged cell cycle, an accumulation at G(2) to M phase transition, and enhanced p53 activity. Interestingly, the impaired M-phase entry and NPC amplification of Wip1-null mice can be reversed in Wip1/p53 double-null mice. Importantly, there is no difference in NPC amplification between p53-null and Wip1/p53 double-null mice. Our data demonstrate that Wip1 regulates the generation of new neural cells in adult olfactory bulb specifically through p53-dependent M-phase entry of the NPC cell cycle.

Induction of PPM1D following DNA-damaging treatments through a conserved p53 response element coincides with a shift in the use of transcription initiation sites.

PPM1D (Wip1), a type PP2C phosphatase, is expressed at low levels in most normal tissues but is overexpressed in several types of cancers. In cells containing wild-type p53, the levels of PPM1D mRNA and protein increase following exposure to genotoxic stress, but the mechanism of regulation by p53 was unknown. PPM1D also has been identified as a CREB-regulated gene due to the presence of a cyclic AMP response element (CRE) in the promoter. Transient transfection and chromatin immunoprecipitation experiments in HCT116 cells were used to characterize a conserved p53 response element located in the 5' untranslated region (UTR) of the PPM1D gene that is required for the p53-dependent induction of transcription from the human PPM1D promoter. CREB binding to the CRE contributes to the regulation of basal expression of PPM1D and directs transcription initiation at upstream sites. Following exposure to ultraviolet (UV) or ionizing radiation, the abundance of transcripts with short 5' UTRs increased in cells containing wild-type p53, indicating increased utilization of downstream transcription initiation sites. In cells containing wild-type p53, exposure to UV resulted in increased PPM1D protein levels even when PPM1D mRNA levels remained constant, indicating post-transcriptional regulation of PPM1D protein levels.

Wip1 phosphatase regulates p53-dependent apoptosis of stem cells and tumorigenesis in the mouse intestine.

Colorectal cancer is one of the major causes of cancer-related deaths. To gain further insights into the mechanisms underlying its development, we investigated the role of Wip1 phosphatase, which is highly expressed in intestinal stem cells, in the mouse model of APC(Min)-driven polyposis. We found that Wip1 removal increased the life span of APC(Min) mice through a significant suppression of polyp formation. This protection was dependent on the p53 tumor suppressor, which plays a putative role in the regulation of apoptosis of intestinal stem cells. Activation of apoptosis in stem cells of Wip1-deficient mice, but not wild-type APC(Min) mice, increased when the Wnt pathway was constitutively activated. We propose, therefore, that the Wip1 phosphatase regulates homeostasis of intestinal stem cells. In turn, Wip1 loss suppresses APC(Min)-driven polyposis by lowering the threshold for p53-dependent apoptosis of stem cells, thus preventing their conversion into tumor-initiating stem cells.

Regulation of ATM/p53-dependent suppression of myc-induced lymphomas by Wip1 phosphatase.

The ataxia telangiectasia mutated (ATM) kinase is a key tumor suppressor that regulates numerous cell cycle checkpoints as well as apoptosis. Here, we report that ATM is a critical player in the regulation of apoptosis and lymphomagenesis in the presence of c-myc. In turn, deletion of the inhibitory ATM phosphatase, Wip1, results in ATM up-regulation and suppression of Emicro-myc-induced B cell lymphomas. Using mouse genetic crosses, we show that the onset of myc-induced lymphomas is dramatically delayed in Wip1-null mice in an ATM- and p53-, but not p38 MAPK- or Arf-, dependent manner. We propose that Wip1 phosphatase is critical for regulating the ATM-mediated tumor surveillance network.

Wip1 phosphatase modulates ATM-dependent signaling pathways.

Deletion of Ppm1d, the gene encoding the Wip1 phosphatase, renders cells resistant to transformation and mice resistant to tumor development. Here, we report that deficiency of Wip1 resulted in activation of the ataxia-telangiectasia mutated (ATM) kinase. In turn, overexpression of Wip1 was sufficient to reduce activation of the ATM-dependent signaling cascade after DNA damage. Wip1 dephosphorylated ATM Ser1981, a site critical for ATM monomerization and activation, and was critical for resetting ATM phosphorylation as cells repaired damaged DNA. We propose that the Wip1 phosphatase is an integral component of an ATM-dependent signaling pathway.