Latency, microenvironment, and the priming of a precancerous senescent cell for malignant transformation

What seems like a good idea can sometimes backfire later. A mechanism that foils cancer in young animals might induce it in older ones, according to a new study. Most mammalian cells, whether in animals or culture dishes, don't divide forever; instead, they eventually undergo cellular senescence, which permanently arrests cell division (see "More Than a Sum of Our Cells" ). In animals, that process might prevent cells with DNA damage or shortened telomeres from growing into tumors (see "Dangerous Liaisons" ). Senescent cells, however, remain in the body--dormant but perhaps not benign. To investigate how senescent cells affect the tissue in which they reside, Krtolica and colleagues grew cultures that contained either presenescent or senescent fibroblasts--cells that compose connective tissue--and then layered test cultures of various epithelial cell types on top of the fibroblasts. The researchers measured proliferation of the epithelial cells by tagging their DNA with fluorescent labels. Senescent, but not presenescent, fibroblasts provoked duplication of cell lines that had previously acquired mutations known to predispose them to cancer, the team found. Neither senescent nor presenescent fibroblasts promoted the growth of normal human epithelial cells. Cell-to-cell contact isn't required for the stimulation: Material secreted by senescent cells induced precancerous cells to grow approximately three times faster than did material secreted by presenescent cells. Senescent cells also appear to promote tumor growth in vivo: Tumors were more prevalent and grew larger in mice injected with precancerous epithelial cells and senescent fibroblasts than in those injected with precancerous cells and presenescent fibroblasts. Senescent cells apparently create a rich environment for tumor growth. This phenomenon, combined with the increased incidence of cancer-causing mutations as time passes, could partially explain the exponential increase in cancer rates as we age. Identifying exactly how senescent cells create fertile ground for cancer might well shed light on how a good deal in youth turns sour with age. --R. John Davenport; suggested by Nick Bishop A. Krtolica, S. Parrinello, S. Lockett, P.-Y. Desprez, J. Campisi, Senescent fibroblasts promote epithelial cell growth and tumorigenesis: A link between cancer and aging. Proc. Natl. Acad. Sci. U.S.A. 98 , 12072-12077 (2001). [Abstract] [Full Text]

Modulating phosphate consumption, a novel therapeutic approach for the control of cancer cell proliferation and tumorigenesis

Heterozygous isocitrate dehydrogenase (IDH) R132H mutation (IDH1R132H/WT) is an early event during gliomagenesis. Clinically, patients with glioma carrying mutant IDH1 respond better to antitumor therapies. However, the mechanism by which IDH1 mutations contribute to gliomagenesis and therapeutic response remains elusive. Here we report that senescence is involved in the improved therapeutic responses of mutant IDH1 glioma cells. Knocking-in IDH1R132H/WT in glioma cells significantly enhanced gliomas cell senescence in response to temozolomide and radiation via a DNA-damage mediated mechanism. We further asked if senescence plays a role in IDH1R132H/WT-induced gliomagenesis. Together with ATRX knockout and p53/RB loss, IDH1R132H/WT transformed nonneoplastic human astroglial cells to form tumors in mouse brains. In-depth characterization revealed that a subset of these precancerous cells underwent senescence-like phenotypic changes, including flat and enlarged-cell morphology, increased senescence marker expression, decreased cell proliferation, and cell-cycle arrest at the G2-M phase. Mechanistic studies indicated that the combination of glioma driver genes (p53/RB/IDH1/ATRX) dramatically increased DNA damage and activated DNAdamage response (DDR) pathways ATR/ATR and Chk1/Chk2 in senescent cells. To determine how senescent cells drive tumor formation, we investigated non-cell-autonomous mechanisms such as senescence-associated secretory phenotype (SASP), a panel of proinflammatory and tissue-remodeling factors implicated in a tumor-permissive microenvironment. We found that astroglial cells carrying p53/RB/ATRX loss and IDH1R132H/WT upregulated key factors in SASP via an epigenetic-mediated mechanism. Our work suggests that drugs that specifically eliminate senescent cells could help kill precancerous cells and senescent tumor cells following antitumor therapies. IMPLICATIONS: The mechanisms by which IDH1 mutations contribute to gliomagenesis and therapeutic responses remain incompletely characterized; this work reveals senescence as a novel mechanism of IDH-mutant-mediated biological impact and describes new therapeutic opportunities concerning IDH1-mutant gliomas.

<div>Abstract<p>Heterozygous isocitrate dehydrogenase (IDH) R132H mutation (IDH1<sup>R132H/WT</sup>) is an early event during gliomagenesis. Clinically, patients with glioma carrying mutant IDH1 respond better to antitumor therapies. However, the mechanism by which IDH1 mutations contribute to gliomagenesis and therapeutic response remains elusive. Here we report that senescence is involved in the improved therapeutic responses of mutant IDH1 glioma cells. Knocking-in IDH1<sup>R132H/WT</sup> in glioma cells significantly enhanced gliomas cell senescence in response to temozolomide and radiation via a DNA-damage mediated mechanism. We further asked if senescence plays a role in IDH1<sup>R132H/WT</sup>-induced gliomagenesis. Together with ATRX knockout and p53/RB loss, IDH1<sup>R132H/WT</sup> transformed nonneoplastic human astroglial cells to form tumors in mouse brains. In-depth characterization revealed that a subset of these precancerous cells underwent senescence-like phenotypic changes, including flat and enlarged-cell morphology, increased senescence marker expression, decreased cell proliferation, and cell-cycle arrest at the G<sub>2</sub>–M phase. Mechanistic studies indicated that the combination of glioma driver genes (p53/RB/IDH1/ATRX) dramatically increased DNA damage and activated DNAdamage response (DDR) pathways ATR/ATR and Chk1/Chk2 in senescent cells. To determine how senescent cells drive tumor formation, we investigated non–cell-autonomous mechanisms such as senescence-associated secretory phenotype (SASP), a panel of proinflammatory and tissue-remodeling factors implicated in a tumor-permissive microenvironment. We found that astroglial cells carrying p53/RB/ATRX loss and IDH1<sup>R132H/WT</sup> upregulated key factors in SASP via an epigenetic-mediated mechanism. Our work suggests that drugs that specifically eliminate senescent cells could help kill precancerous cells and senescent tumor cells following antitumor therapies.</p>Implications:<p>The mechanisms by which IDH1 mutations contribute to gliomagenesis and therapeutic responses remain incompletely characterized; this work reveals senescence as a novel mechanism of IDH-mutant–mediated biological impact and describes new therapeutic opportunities concerning IDH1-mutant gliomas.</p></div>

Hepatocellular carcinoma (HCC) is a malignant tumor associated with a generally poor prognosis and a high rate of recurrence. HCC usually develops in the context of chronic liver diseases, and long-lasting premalignant conditions precede cancer development. A promising therapeutic approach is to eliminate precancerous cells, which are considered as the precursors of cancer stem cells, to prevent further malignant transformation. In this study, we identified a subpopulation of precancerous cells in a rat liver carcinogenesis model, which were enriched in CD133(+)CD44(+)CD45(-)HIS49(-) cells that formed part of the hepatic oval cells fraction. Prospective isolation of the precancerous cells using flow cytometry identified stem cell properties such as the ability to expand clonally and differentiate into bi-lineage cell types. Furthermore, an acyclic retinoid, which was recently shown to improve overall survival after HCC resection, directly inhibited the extensive expansion of the isolated precancerous cells in vitro and decreased the emergence of the precancerous cells and their progeny in vivo. Long-term follow-up after the acyclic retinoid treatment confirmed reduction in precancerous changes, ultimately resulting in suppression of HCC development. These findings, together with data from recent clinical trials showing marked reduction in intrahepatic recurrence, suggest that acyclic retinoid directly prevents de novo HCC by inhibiting the development of precancerous cells. Given recent advances in diagnostic techniques and the establishment of surveillance programs, the targeting of precancerous cells may have a huge impact on preventative cancer therapies.

Dissecting primary and secondary senescence to enable new senotherapeutic strategies

Tumor cell formation by normal somatic cells fusing and cancer prevention prospects

Malignant cells are known to contain high amounts of glutathione, a potent endogenous anti-oxidant, which is believed to contribute to their increased rate of mitosis and resistance to chemo and radiotherapies. But there is very little information as to the levels of glutathione in precancerous cells. In the present study, it was noted that the concentrations of total glutathione is significantly higher in human precancerous cells compared to cancerous cells. The higher amounts of glutathione noted in precancerous cells could be a protective mechanism adopted to prevent oxidative stress-induced DNA damage that aids malignant transformation process.

PurposeSenescence is a terminal growth arrest that functions as a tumor suppressor in aging and precancerous cells and is a response to selected anticancer compounds. Lysosomal-β-galactosidase (GLB1) hydrolyzes β-galactose from glycoconjugates and is the origin of senescence-associated β-gal activity (SA-β-gal). Using a new GLB1 antibody, senescence biology was investigated in prostate cancer (PCa) tissues.Experimental Design In vitro characterization of GLB1 was determined in primary prostate epithelial cell cultures passaged to replicative senescence and in therapy-induced senescence in PCa lines using chemotherapeutic agents. FFPE tissue microarrays were subjected to immunofluorescent staining for GLB1, Ki67 and HP1γ and automated quantitative imaging initially using AQUA in exploratory samples and Vectra in a validation series.ResultsGLB1 expression accumulates in replicative and induced senescence and correlates with senescent morphology and P16 (CDKN2) expression. In tissue arrays, quantitative imaging detects increased GLB1 expression in high-grade prostatic intraepithelial neoplasia (HGPIN), known to contain senescent cells, and cancer compared to benign prostate tissues (p<0.01) and senescent cells contain low Ki67 and elevated HP1γ. Within primary tumors, elevated GLB1 associates with lower T stage (p=0.01), localized versus metastatic disease (p=0.0003) and improved PSA-free survival (p=0.03). Increased GLB1 stratifies better PSA-free survival in intermediate grade PCa (0.01). Tissues that elaborate higher GLB1 display increased uniformity of expression.ConclusionIncreased GLB1 is a valuable marker in formalin-fixed paraffin-embedded (FFPE) tissues for the senescence-like phenotype and associates with improved cancer outcomes. This protein addresses a lack of senescence markers and should be applicable to study the biologic role of senescence in other cancers.

Autophagy has recently been implicated in both the prevention and progression of cancer. However, the molecular basis for the relationship between autophagy induction and the initial acquisition of malignancy is currently unknown. Here, we provide the first evidence that autophagy is essential for oncogenic K-Ras (K-RasV12)-induced malignant cell transformation. Retroviral expression of K-RasV12 induced autophagic vacuole formation and malignant transformation in human breast epithelial cells. Interestingly, pharmacological inhibition of autophagy completely blocked K-RasV12-induced, anchorage-independent cell growth on soft agar. Both mRNA and protein levels of ATG5 and ATG7 (autophagy-specific genes 5 and 7, respectively) were increased in cells overexpressing K-RasV12. Targeted suppression of ATG5 or ATG7 expression by short hairpin (sh) RNA inhibited cell growth on soft agar and tumor formation in nude mice. Moreover, inhibition of reactive oxygen species (ROS) with antioxidants clearly attenuated K-RasV12-induced ATG5 and ATG7 induction, autophagy, and malignant cell transformation. MAPK pathway components were activated in cells overexpressing K-RasV12, and inhibition of JNK blunted induction of ATG5 and ATG7 and subsequent autophagy. In addition, pretreatment with antioxidants completely inhibited K-RasV12-induced JNK activation. Our results provide novel evidence that autophagy is critically involved in malignant transformation by oncogenic K-Ras and show that reactive oxygen species-mediated JNK activation plays a causal role in autophagy induction through up-regulation of ATG5 and ATG7.

Testicular germ cell tumors are in most cases preceded by an intratubular noninvasive form of neoplasia, known as carcinoma in situ (CIS, Skakkebaek and Berthelsen 1978). These atypical germ cells resemble gonocytes or spermatogonia (Nüesch-Bachmann and Hedinger 1977), but also show nuclear atypia indicative of malignant transformation. Atypical intratubular cells have been known to precede overt tumors for up to 16 years (Bannwart et al. 1988). However, they do not have invasive properties and the attempts to grow them as xenografts in nude mice have been unsuccessful (Walt and Stevens unpublished results). The transition of CIS to invasive carcinoma therefore remains poorly understood. In an effort to understand better the pathobiology of testicular neoplasias and their precancerous precursors, we have studied the expression of certain biochemical tumor markers (Walt et al. 1986) and of alkaline phosphatase isozymes (Hofmann et al. 1989), the activity of nucleolus organizer regions (NORs) (Delozier-Blanchet et al. 1986a), and the presence of a number of specific chromosomal regions by in situ hybridization on paraffin sections (Emmerich et al. 1989). For these investigations we have used xenografts and their related cell lines (DelozierBlanchet et al. 1986b; Emmerich et al. 1989; Hofmann et al. 1989; Walt et al. 1986) as well as primary tumors and primary precancerous and ejaculated germ cells (Jauch et al. 1989; Walt et al. 1989). We hope that this summary of data will allow the development of new strategies for further research in this area.KeywordsGerm CellGerm Cell TumorEmbryonal CarcinomaTesticular Germ Cell TumorTesticular Germ CellThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Expression and role of methyltransferase 3 in oral malignant transformation

Oral squamous cell carcinoma (OSCC) is usually diagnosed at late stages, which leads to high morbidity. There are evidence that chronic inflammation (eg oral lichen planus [OLP]) was a risk factor of OSCC, but often misdiagnosed or ignored until invasion and metastasis. By applying precision medicine, the molecular microenvironment variations and relevant biomarkers for the malignant transformation from OLP to OSCC can be fully investigated. Several studies pointed out that the metabolic pathway were suppressed in OSCC. However, it remains unclear how the systemic profile of the metabolites change during the malignant transformation. In this study, we examined and compared the mucosa samples from 11 healthy individuals, 10 OLP patients and 21 OSCC patients. Based on the results, succinate, a key metabolite of the tricarboxylic acid cycle pathway, was accumulated in the primary cultured precancerous OLP keratinocytes and OSCC cells. Then, we found that succinate activated the hypoxia‐inducible factor‐1 alpha (HIF‐1α) pathway and induced apoptosis, which could also be up‐regulated by the tumour suppressor lncRNA MEG3. These results suggested the critical roles of succinate and MEG3 in the metabolic changes during malignant transformation from OLP to OSCC, which indicated that succinate, HIF1α and downstream proteins might serve as new biomarkers of precancerous OLP for early diagnosis and therapeutic monitoring. In addition, succinate or its prodrugs might become a potential therapy for the prevention or treatment of OSCC.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. The lack of effective targeted drugs has become a challenge on treating HCC patients. Cellular senescence is closely linked to the occurrence, development, and therapy of tumor. Induction of cellular senescence and further activation of immune surveillance provides a new strategy to develop HCC targeted drugs, that is, senescence-induced therapy for HCC. Precancerous hepatocytes or HCC cells can be induced into senescent cells, subsequently producing senescence-associated secretory phenotype (SASP) factors. SASP factors recruit and activate various types of immune cells, including T cells, NK cells, macrophages, and their subtypes, which carry out the role of immune surveillance and elimination of senescent cells, ultimately preventing the occurrence of HCC or inhibiting the progression of HCC. Specific interventions in several checkpoints of senescence-mediated therapy will make positive contributions to suppress tumorigenesis and progression of HCC, for instance, by applying small molecular compounds to induce cellular senescence or selecting cytokines/chemokines to activate immunosurveillance, supplementing adoptive immunocytes to remove senescent cells, and screening chemical drugs to induce apoptosis of senescent cells or accelerate clearance of senescent cells. These interventional checkpoints become potential chemotherapeutic targets in senescence-induced therapy for HCC. In this review, we focus on the frontiers of senescence-induced therapy and discuss senescent characteristics of hepatocytes during hepatocarcinogenesis as well as the roles and mechanisms of senescent cell induction and clearance, and cellular senescence-related immunosurveillance during the formation and progression of HCC.

The cell cycle is a redox cycle: Linking phase-specific targets to cell fate