Compounds with specific cytotoxic activity in senescent cells, or senolytics, support the causal involvement of senescence in aging and offer therapeutic interventions. Here we report the ...identification of Cardiac Glycosides (CGs) as a family of compounds with senolytic activity. CGs, by targeting the Na+/K+ATPase pump, cause a disbalanced electrochemical gradient within the cell causing depolarization and acidification. Senescent cells present a slightly depolarized plasma membrane and higher concentrations of H+, making them more susceptible to the action of CGs. These vulnerabilities can be exploited for therapeutic purposes as evidenced by the in vivo eradication of tumors xenografted in mice after treatment with the combination of a senogenic and a senolytic drug. The senolytic effect of CGs is also effective in the elimination of senescence-induced lung fibrosis. This experimental approach allows the identification of compounds with senolytic activity that could potentially be used to develop effective treatments against age-related diseases.
Genetic data indicate that abrogation of Notch-Rbpj or Wnt-β-catenin pathways results in the loss of the intestinal stem cells (ISCs). However, whether the effect of Notch is direct or due to the ...aberrant differentiation of the transit-amplifying cells into post-mitotic goblet cells is unknown. To address this issue, we have generated composite tamoxifen-inducible intestine-specific genetic mouse models and analyzed the expression of intestinal differentiation markers. Importantly, we found that activation of β-catenin partially rescues the differentiation phenotype of Rbpj deletion mutants, but not the loss of the ISC compartment. Moreover, we identified Bmi1, which is expressed in the ISC and progenitor compartments, as a gene that is co-regulated by Notch and β-catenin. Loss of Bmi1 resulted in reduced proliferation in the ISC compartment accompanied by p16(INK4a) and p19(ARF) (splice variants of Cdkn2a) accumulation, and increased differentiation to the post-mitotic goblet cell lineage that partially mimics Notch loss-of-function defects. Finally, we provide evidence that Bmi1 contributes to ISC self-renewal.
Notch has been linked to β-catenin-dependent tumorigenesis; however, the mechanisms leading to Notch activation and the contribution of the Notch pathway to colorectal cancer is not yet understood. ...By microarray analysis, we have identified a group of genes downstream of Wnt/β-catenin (down-regulated when blocking Wnt/β-catenin) that are directly regulated by Notch (repressed by γ-secretase inhibitors and up-regulated by active Notch1 in the absence of β-catenin signaling). We demonstrate that Notch is downstream of Wnt in colorectal cancer cells through β-catenin-mediated transcriptional activation of the Notch-ligand Jagged1. Consistently, expression of activated Notch1 partially reverts the effects of blocking Wnt/β-catenin pathway in tumors implanted s.c. in nude mice. Crossing APCMin/⁺ with Jagged1⁺/Δ mice is sufficient to significantly reduce the size of the polyps arising in the APC mutant background indicating that Notch is an essential modulator of tumorigenesis induced by nuclear β-catenin. We show that this mechanism is operating in human tumors from Familial Adenomatous Polyposis patients. We conclude that Notch activation, accomplished by β-catenin-mediated up-regulation of Jagged1, is required for tumorigenesis in the intestine. The Notch-specific genetic signature is sufficient to block differentiation and promote vasculogenesis in tumors whereas proliferation depends on both pathways.
The hierarchical relationships between stem cells, lineage-committed progenitors, and differentiated cells remain unclear in several tissues, due to a high degree of cell plasticity, allowing cells ...to switch between different cell states. The mouse mammary gland, similarly to other tissues such as the prostate, the sweat gland, and the respiratory tract airways, consists of an epithelium exclusively maintained by unipotent progenitors throughout adulthood. Such unipotent progenitors, however, retain a remarkable cellular plasticity, as they can revert to multipotency during epithelial regeneration as well as upon oncogene activation. Here, we revise the current knowledge on mammary cell hierarchies in light of the most recent lineage tracing studies performed in the mammary gland and highlight how stem cell differentiation or reversion to multipotency are at the base of tumor development and progression. In addition, we will discuss the current knowledge about the interplay between tumor cells of origin and defined genetic mutations, leading to different tumor types, and its implications in choosing specific therapeutic protocols for breast cancer patients.
Delta ligands regulate Notch signaling in normal intestinal stem cells, while Jagged1 activates Notch in intestinal adenomas carrying active β-catenin. We used the Apc
mouse model, tumor spheroid ...cultures, and patient-derived orthoxenografts to address this divergent ligand-dependent Notch function and its implication in disease. We found that intestinal-specific Jag1 deletion or antibody targeting Jag1 prevents tumor initiation in mice. Addiction to Jag1 is concomitant with the absence of Manic Fringe (MFNG) in adenoma cells, and its ectopic expression reverts Jag1 dependence. In 239 human colorectal cancer patient samples, MFNG imposes a negative correlation between Jag1 and Notch, being high Jag1 in the absence of MFNG predictive of poor prognosis. Jag1 antibody treatment reduces patient-derived tumor orthoxenograft growth without affecting normal intestinal mucosa. Our data provide an explanation to Jag1 dependence in cancer, and reveal that Jag1-Notch1 interference provides therapeutic benefit in a subset of colorectal cancer and FAP syndrome patients.
Antibody-drug conjugates (ADC) are antineoplastic agents recently introduced into the antitumor arsenal. T-DM1, a trastuzumab-based ADC that relies on lysosomal processing to release the payload, is ...approved for HER2-positive breast cancer. Next-generation ADCs targeting HER2, such as vic-trastuzumab duocarmazine (SYD985), bear linkers cleavable by lysosomal proteases and membrane-permeable drugs, mediating a bystander effect by which neighboring antigen-negative cells are eliminated. Many antitumor therapies, like DNA-damaging agents or CDK4/6 inhibitors, can induce senescence, a cellular state characterized by stable cell-cycle arrest. Another hallmark of cellular senescence is the enlargement of the lysosomal compartment. Given the relevance of the lysosome to the mechanism of action of ADCs, we hypothesized that therapies that induce senescence would potentiate the efficacy of HER2-targeting ADCs. Treatment with the DNA-damaging agent doxorubicin and CDK4/6 inhibitor induced lysosomal enlargement and senescence in several breast cancer cell lines. While senescence-inducing drugs did not increase the cytotoxic effect of ADCs on target cells, the bystander effect was enhanced when HER2-negative cells were cocultured with HER2-low cells. Knockdown experiments demonstrated the importance of cathepsin B in the enhanced bystander effect, suggesting that cathepsin B mediates linker cleavage. In breast cancer patient-derived xenografts, a combination treatment of CDK4/6 inhibitor and SYD985 showed improved antitumor effects over either treatment alone. These data support the strategy of combining next-generation ADCs targeting HER2 with senescence-inducing therapies for tumors with heterogenous and low HER2 expression.
Combining ADCs against HER2-positive breast cancers with therapies that induce cellular senescence may improve their therapeutic efficacy by facilitating a bystander effect against antigen-negative tumor cells.
Lineage tracing studies have become a well-suited approach to reveal cellular hierarchies and tumor heterogeneity. Cellular heterogeneity, particularly in breast cancer, is still one of the main ...concerns regarding tumor progression and resistance to anti-cancer therapies. Here, we review the current knowledge about lineage tracing analyses that have contributed to an improved comprehension of the complexity of mammary tumors, highlighting how targeting different mammary epithelial cells and tracing their progeny can be useful to explore the intra- and inter-heterogeneity observed in breast cancer. In addition, we examine the strategies used to identify the cell of origin in different breast cancer subtypes and summarize how cellular plasticity plays an important role during tumorigenesis. Finally, we evaluate the clinical implications of lineage tracing studies and the challenges remaining to address tumor heterogeneity in breast cancer.
Lineage tracing is now considered the gold standard approach to study cellular hierarchies and cell fate in vivo (McKenna and Gagnon, Development 146:dev169730, 2019; Kretzschmar and Watt, Cell ...148:33-45, 2012). This type of clonal analysis consists of genetically labeling defined cells and following their destiny and progeny in vivo and in situ.Here we will describe different existing in vivo systems to clonally trace targeted cells and will discuss their respective advantages and inconveniences; we will then provide stepwise instructions for setting up and evaluate lineage tracing experiments, listing the most common downstream analyses and read-out assays.
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