Senescence Escape Research Articles

Anterior gradient protein 2 is a marker of tumor aggressiveness in breast cancer and favors chemotherapy‑induced senescence escape.

Among the different chemotherapies available, genotoxic drugs are widely used. In response to these drugs, particularly doxorubicin, tumor cells can enter into senescence. Chemotherapy-induced senescence (CIS) is a complex response. Long described as a definitive arrest of cell proliferation, the present authors and various groups have shown that this state may not be complete and could allow certain cells to reproliferate. The mechanism could be due to the activation of new signaling pathways. In the laboratory, the proteins involved in these pathways and triggering cell proliferation were studied. The present study determined a new role for anterior gradient protein 2 (AGR2) in vivo in patients and in vitro in a senescence escape model. AGR2's implication in breast cancer patients and proliferation of senescent cells was assessed based on a SWATH-MS proteomic study of patients' samples and RNA interference technology on cell lines. First, AGR2 was identified and it was found that its concentration is higher in the serum of patients with breast cancer and that this high concentration is associated with metastasis occurrence. An inverse correlation between intratumoral AGR2 expression and the senescence marker p16 was also observed. This observation led to the study of the role of AGR2 in the CIS escape model. In this model, it was found that AGR2 is overexpressed in cells during senescence escape and that its loss considerably reduces this phenomenon. Furthermore, it was shown that the extracellular form of AGR2 stimulated the reproliferation of senescent cells. The power of proteomic analysis based on the SWATH-MS approach allowed the present study to highlight the mammalian target of rapamycin (mTOR)/AKT signaling pathway in the senescence escape mechanism mediated by AGR2. Analysis of the two signaling pathways revealed that AGR2 modulated RICTOR and AKT phosphorylation. All these results showed that AGR2 expression in sera and tumors of breast cancer patients is a marker of tumor progression and metastasis occurrence. They also showed that its overexpression regulates CIS escape via activation of the mTOR/AKT signaling pathway.

Cancer Stem Cells-Origins and Biomarkers: Perspectives for Targeted Personalized Therapies.

The use of biomarkers in diagnosis, therapy and prognosis has gained increasing interest over the last decades. In particular, the analysis of biomarkers in cancer patients within the pre- and post-therapeutic period is required to identify several types of cells, which carry a risk for a disease progression and subsequent post-therapeutic relapse. Cancer stem cells (CSCs) are a subpopulation of tumor cells that can drive tumor initiation and can cause relapses. At the time point of tumor initiation, CSCs originate from either differentiated cells or adult tissue resident stem cells. Due to their importance, several biomarkers that characterize CSCs have been identified and correlated to diagnosis, therapy and prognosis. However, CSCs have been shown to display a high plasticity, which changes their phenotypic and functional appearance. Such changes are induced by chemo- and radiotherapeutics as well as senescent tumor cells, which cause alterations in the tumor microenvironment. Induction of senescence causes tumor shrinkage by modulating an anti-tumorigenic environment in which tumor cells undergo growth arrest and immune cells are attracted. Besides these positive effects after therapy, senescence can also have negative effects displayed post-therapeutically. These unfavorable effects can directly promote cancer stemness by increasing CSC plasticity phenotypes, by activating stemness pathways in non-CSCs, as well as by promoting senescence escape and subsequent activation of stemness pathways. At the end, all these effects can lead to tumor relapse and metastasis. This review provides an overview of the most frequently used CSC markers and their implementation as biomarkers by focussing on deadliest solid (lung, stomach, liver, breast and colorectal cancers) and hematological (acute myeloid leukemia, chronic myeloid leukemia) cancers. Furthermore, it gives examples on how the CSC markers might be influenced by therapeutics, such as chemo- and radiotherapy, and the tumor microenvironment. It points out, that it is crucial to identify and monitor residual CSCs, senescent tumor cells, and the pro-tumorigenic senescence-associated secretory phenotype in a therapy follow-up using specific biomarkers. As a future perspective, a targeted immune-mediated strategy using chimeric antigen receptor based approaches for the removal of remaining chemotherapy-resistant cells as well as CSCs in a personalized therapeutic approach are discussed.

The many types of heterogeneity in replicative senescence.

Replicative senescence, which is induced by telomere shortening, underlies the loss of regeneration capacity of organs and is ultimately detrimental to the organism. At the same time, it is required to protect organisms from unlimited cell proliferation that may arise from numerous stimuli or deregulations. One important feature of replicative senescence is its high level of heterogeneity and asynchrony, which promote genome instability and senescence escape. Characterizing this heterogeneity and investigating its sources are thus critical to understanding the robustness of replicative senescence. Here we review the different aspects of senescence driven by telomere attrition that are subject to variation in Saccharomyces cerevisiae, the current understanding of the molecular processes at play, and the consequences of heterogeneity in replicative senescence.

Telomere elongation via alternative lengthening of telomeres (ALT) and telomerase activation in primary metastatic medulloblastoma of childhood.

Elongation of telomeres is necessary for tumor cell immortalization and senescence escape; neoplastic cells use to alternative pathways to elongate telomeres: telomerase reactivation or a telomerase-independent mechanism termed alternative lengthening of telomeres (ALT). Telomerase and ALT pathway has been explored in adult and pediatric gliomas and medulloblastomas (MDBs); however, these mechanisms were not previously investigated in MDBs metastatic at the onset. Therefore, we analyzed the activation of telomerase and ALT pathway in a homogenous cohort of 43 pediatric metastatic medulloblastomas, to investigate whether telomere elongation could play a role in the biology of metastatic MDB. We evaluated telomeres length via telomere-specific fluorescence in situ hybridization (Telo-FISH); we assessed nuclear expression of ATRX by immunohistochemistry (IHC). H3F3A and TERT promoter mutations were analyzed by pyrosequencing, while UTSS methylation status was analyzed via methylation-specific-PCR (MS-PCR). H3F3A mutations were absent in all MDBs, 30% of samples showed ATRX nuclear loss, 18.2% of cases were characterized by TERT promoter mutations, while 60.9% harboured TERT promoter hyper-methylation in the UTSS region. Elongation of telomeres was found in 42.8% of cases. Metastatic MDBs control telomere elongation via telomerase activation (10.7%), induced by TERT promoter mutations in association with UTSS hyper-methylation, and ALT mechanism (32.1%), triggered by ATRX inactivation. Among non-metastatic MDBs, only 5.9% (1/17) showed ATRX nuclear loss with activation of ALT. Our metastatic cases frequently activate ALT pathway, suggesting that it is a common process for senescence escape in primary metastatic medulloblastomas. Furthermore, the activation of mechanisms for telomere elongation is not restricted to certain molecular subgroups in this high-risk group of MDBs.

Regulation of senescence escape by TSP1 and CD47 following chemotherapy treatment

Senescence is a tumor-suppressive mechanism induced by telomere shortening, oncogenes, or chemotherapy treatment. Although it is clear that this suppressive pathway leads to a permanent arrest in primary cells, this might not be the case in cancer cells that have inactivated their suppressive pathways. We have recently shown that subpopulations of cells can escape chemotherapy-mediated senescence and emerge as more transformed cells that induce tumor formation, resist anoikis, and are more invasive. In this study, we characterized this emergence and showed that senescent cells favor tumor growth and metastasis, in vitro and in vivo. Senescence escape was regulated by secreted proteins produced during emergence. Among these, we identified thrombospondin-1 (TSP1), a protein produced by senescent cells that prevented senescence escape. Using SWATH quantitative proteomic analysis, we found that TSP1 can be detected in the serum of patients suffering from triple-negative breast cancer and that its low expression was associated with treatment failure. The results also indicate that senescence escape is explained by the emergence of CD47low cells that express a reduced level of CD47, the TSP1 receptor. The results show that CD47 expression is regulated by p21waf1. The cell cycle inhibitor was sufficient to maintain senescence since its downregulation in senescent cells increased cell emergence. This leads to the upregulation of Myc, which then binds to the CD47 promoter to repress its expression, allowing the generation of CD47low cells that escape the suppressive arrest. Altogether, these results uncovered a new function for TSP1 and CD47 in the control of chemotherapy-mediated senescence.

Regulation of senescence escape by the cdk4\u2013EZH2\u2013AP2M1 pathway in response to chemotherapy

Senescence is a tumor suppressive mechanism that induces a permanent proliferative arrest in response to an oncogenic insult or to the genotoxic stress induced by chemotherapy. We have recently described that some cells can escape this arrest, either because senescence was incomplete or as a consequence of a phenotypic adaptation. Malignant cells which resisted senescence emerged as more transformed cells that resist anoikis and rely on survival pathways activated by Akt and Mcl-1. In this study, we further characterize senescence escape, investigating how emergent cells could reproliferate. During the initial step of chemotherapy-induced senescence (CIS), we found that cyclin D1 was upregulated and that cell emergence was prevented when its main partner cdk4 was inactivated. Results indicate that this kinase induced the upregulation of the EZH2 methylase, a component of the polycomb PRC2 complex. Downregulated during the early step of treatment, the methylase was reactivated in clones that escaped senescence. The inactivation of EZH2, either by siRNA or by specific inhibitors, led to a specific inhibition of cell emergence. We used quantitative proteomic analysis to identify new targets of the methylase involved in senescence escape. We identified proteins involved in receptor endocytosis and described new functions for the AP2M1 protein in the control of chemotherapy-mediated senescence. Our results indicate that AP2M1 is involved in the transmission of secreted signals produced by senescent cells, suggesting that this pathway might regulate specific receptors involved in the control of CIS escape. In light of these results, we therefore propose that the cdk4–EZH2–AP2M1 pathway plays an important role during chemotherapy resistance and senescence escape. Since targeted therapies are available against these proteins, we propose that they should be tested in the treatment of colorectal or breast cancers that become resistant to first-line genotoxic therapies.

Dedifferentiation into blastomere-like cancer stem cells via formation of polyploid giant cancer cells

Our recent perplexing findings that polyploid giant cancer cells (PGCCs) acquired embryonic-like stemness and were capable of tumor initiation raised two important unanswered questions: how do PGCCs acquire such stemness, and to which stage of normal development do PGCCs correspond. Intriguingly, formation of giant cells due to failed mitosis/cytokinesis is common in the blastomere stage of the preimplantation embryo. However, the relationship between PGCCs and giant blastomeres has never been studied. Here, we tracked the fate of single PGCCs following paclitaxel-induced mitotic failure. Morphologically, early spheroids derived from PGCCs were indistinguishable from human embryos at the blastomere, polyploid blastomere, compaction, morula and blastocyst-like stages by light, scanning electron or three-dimensional confocal scanning microscopy. Formation of PGCCs was associated with activation of senescence, while budding of daughter cells was associated with senescence escape. PGCCs showed time- and space-dependent activation of expression of the embryonic stem cell markers OCT4, NANOG, SOX2 and SSEA1 and lacked expression of Xist. PGCCs acquired mesenchymal phenotype and were capable of differentiation into all three germ layers in vitro. The embryonic-like stemness of PGCCs was associated with nuclear accumulation of YAP, a key mediator of the Hippo pathway. Spheroids derived from single PGCCs grew into a wide spectrum of human neoplasms, including germ cell tumors, high-grade and low-grade carcinomas and benign tissues. Daughter cells derived from PGCCs showed attenuated capacity for invasion and increased resistance to paclitaxel. We also observed formation of PGCCs and dedifferentiation in ovarian cancer specimens from patients treated with chemotherapy. Taken together, our findings demonstrate that PGCCs represent somatic equivalents of blastomeres, the most primitive cancer stem cells reported to date. Thus, our studies reveal an evolutionarily conserved archaic embryonic program in somatic cells that can be de-repressed for oncogenesis. Our work offers a new paradigm for cancer origin and disease relapse.

Abstract 1054: SNAI1 regulates the hexosamine biosynthetic pathway to promote tumorigenesis and oncogene-induced senescence escape in lung cancer

Abstract Lung cancer is the deadliest and most common malignancy worldwide. Tumor invasion and metastasis influence the dismal 16% overall 5-year survival. Evidence suggests malignancies commandeer epithelial plasticity programs like the epithelial-mesenchymal transition (EMT) to promote tumorigenesis, tumor progression, and metastasis. Furthermore, EMT has recently been linked to alterations in cancer metabolism, which can engender adaptation to environmental stress while supporting the macromolecular demand of rapid proliferation. Thus it is important to better understand the emerging roles of EMT in regulating cancer metabolism. SNAI1 is a master EMT regulator and is aberrantly overexpressed in many cancers including lung cancer. To investigate the role of SNAI1 in tumorigenesis, we created a novel inducible autochthonous mouse model. Our model reveals SNAI1 overexpressing mutant Kras mice generate identifiable tumors twice as fast (median = 14 weeks) as mice overexpressing mutant Kras alone (median = 30 weeks). To identify possible mechanisms of SNAI1-accelerated tumorigenesis, we used mRNA expression profiling. We identified significantly altered gene signatures related to metabolic reprogramming. Specifically, SNAI1 upregulates the hexosamine biosynthetic pathway (HBP) in mutant Kras-driven mouse lung tumors. SNAI1 elevates protein and mRNA of the rate limiting step (GFPT2) and final step (UAP1) of this pathway. This SNAI1-driven promotion of the HBP was recapitulated in vitro using human non-small cell lung cancer (NSCLC) SNAI1 isogenic cell lines. NSCLC mRNA expression data from The Cancer Genome Atlas (TCGA) also showed SNAI1 expression in patient tumor samples correlate with a significant tendency towards co-expression with HBP genes (Fisher's exact test, p<0.05). Functionally, the final metabolite generated by the HBP, UDP-GlcNAc, is an essential nucleotide sugar found in a plethora of N- and O-linked glycosylations, regulating protein stability and activity. Here we show SNAI1 promotes flux through the HBP to promote increased total levels of O-GlcNAcylated proteins in lung tumors in vivo. In so doing, modulation of the HBP and specific glycosylations may impact cell death and senescence, suggesting a novel targetable EMT-HBP axis. Numerous oncogenes, including c-Myc and SNAI1 itself, are known to be stabilized by O-GlcNAcylation. This link is becoming of increasing importance in cancer biology, as half of the body of literature on O-GlcNAcylation and cancer has been published in the past three years. Together, our data suggest heightened protein O-GlcNAcylation, mediated by SNAI1 and the HBP, serves as a novel means by which EMT contributes to mutant Kras-induced lung tumorigenesis and tumor progression. Citation Format: Kekoa Taparra, Hailun Wang, Katriana Nugent, Reem Malek, Jen Groves, Gokben Yildirir, Brian Simons, Dean Felsher, Natasha Zachara, Phuoc Tran. SNAI1 regulates the hexosamine biosynthetic pathway to promote tumorigenesis and oncogene-induced senescence escape in lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1054.

A novel role for DNA single-strand breaks in senescence and neoplastic escape of epithelial cells

ABSTRACTIn contrast to fibroblasts, epithelial cells spontaneously escape from senescence and develop clones of mutated, transformed, and tumorigenic cells. Recently, we revealed that accumulation of unrepaired DNA single-strand breaks is a trigger of the p16 (CDKN2)-dependent cell cycle arrest pathway in senescent epithelial cells and also the mutagenic motor of post-senescence neoplastic escape.

Combination with γ-secretase inhibitor prolongs treatment efficacy of BRAF inhibitor in BRAF-mutated melanoma cells

Oncogenic triggering of the MAPK pathway in melanocytes results in senescence, and senescence escape is considered as one critical step for melanocytic transformation. In melanoma, induction of a senescent-like state by BRAF-inhibitors (BRAFi) in a fraction of treated cells – instead of killing – contributes to the repression of tumor growth, but may also provide a source for relapse. Here, we demonstrate that NOTCH activation in melanocytes is not only growth-promoting but it also protects these cells against oncogene-induced senescence. In turn, treatment of melanoma cells with an inhibitor of the NOTCH-activating enzyme γ-secretase led to induction of a senescent-like status in a fraction of the cells but overall achieved only a moderate inhibition of melanoma cell growth. However, combination of γ-secretase inhibitor (GSI) with BRAFi markedly increased the treatment efficacy particularly in long-term culture. Moreover, even melanoma cells starting to regrow after continuous BRAFi treatment – the major problem of BRAFi therapy in patients – can still be affected by the combination treatment. Thus, combining GSI with BRAFi increases the therapeutic efficacy by, at least partially, prolonging the senescent-like state of treated cells.

Non-Cell-Autonomous Regulation of Cellular Senescence in Cancer.

Cellular senescence is a permanent growth arrest that is broadly recognized to act as a barrier against tumorigenesis. Senescence is predominant in premalignant tumors, and senescence escape is thought to be required for tumor progression. Importantly, evidences indicate that cell-autonomous mechanisms, such as genetic alterations or therapeutic interventions targeting specific genetic pathways, can affect the senescence response in cancer. Nevertheless, new findings have emerged in the last few years that indicate a fundamental role for the tumor microenvironment in the regulation of cellular senescence. Indeed, cytokines belonging to the senescent secretome, as well as tumor-infiltrating immune subsets, have been described to modulate the senescence response in tumors. Such evidence demonstrates that senescence initiation also relies on non-cell-autonomous mechanisms, which are discussed in the present review.

Abstract 1033: Alternative tumor suppressor mechanisms in lung carcinomas - modulation of senescence by growth factor-induced signaling

Abstract Cellular signaling pathways integrate multiple intra- and extracellular signals and thereby influence cell fate. Outcome of oncogene-targeted pharmacotherapies (cell cycle inhibition, apoptosis, senescence, autophagy) depends on variable factors (e.g. compound, dose, duration, cellular genetic background etc.). Oncogene-induced senescence is recognized as an important tumor-suppressive mechanism, bypassing of which may contribute to malignant transformation of the cells. On the other hand, senescence induction, in addition to apoptosis stimulation and proliferation suppression, is responsible for the cytostatic activity of many commonly used anticancer drugs. Therefore, senescence escape must be considered as an important drug-resistance factor. In our work we characterize,,DNA Damage-independent” senescence induced by tyrosine kinase inhibitors (TKI) and growth factor deprivation of non-small cell lung cancer (NSCLC) cell lines and other EGFR-dependent cells. Treatment of different cell lines with various concentrations of EGFR-TKIs was followed by the assessment of cell fate (i.e. apoptosis, cell cycle distribution, senescence and clonogenic survival) as well as intracellular signaling. We could show that treatment of EGFR-dependent cells with specific EGF receptor tyrosine kinase inhibitors (EGFR-TKI) can lead to generation of reactive oxygen species (ROS), activation of a DNA damage response (DDR) program and cellular senescence. Surprisingly, under certain experimental conditions DDR activation was found to be independent from detectable DNA damage. Key players of DDR signaling were shown to be essential for senescence induction. Molecular dissection of crucial signaling nodes and molecular switches involved in modulation of cell fate in oncogene dependent cells treated with specific EGFR-inhibitors is currently being performed. Furthermore, our work is focussed on the identification and characterization of cellular factors activating DNA damage response (DDR) pathways in the absence of DNA damage under certain experimental conditions. Understanding the interplay between theses decision making pathways either leading to proliferative arrest, cellular senescence or apoptosis will help improve current pharmacotherapies and develop new therapeutic approaches. Note: This abstract was not presented at the meeting. Citation Format: Boyka Markova, Frank Breitenbuecher, Martin Schuler. Alternative tumor suppressor mechanisms in lung carcinomas - modulation of senescence by growth factor-induced signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1033. doi:10.1158/1538-7445.AM2015-1033

Multidrug resistance protein 3 loss promotes tumor formation by inducing senescence escape.

Oncogenic-stress-induced senescence (OIS) is a stress response allowing normal cells, when receiving oncogenic signals, to stably arrest their proliferation. OIS thus acts to prevent aberrant cell proliferation and tumor formation. To identify novel tumor suppressive pathways, we have recently completed a loss-of-function genetic screen to identify novel genes promoting escape from OIS and thus, potentially, tumor formation when their functions are lost. Using this approach, we unexpectedly found that loss of function of the multidrug resistance protein 3 (MRP3 or ABCC3) promotes escape from OIS in human epithelial cells. Importantly, ABCC3 expression is reduced in human skin tumors, and ABCC3-knockout mice display increased sensitivity to RAS-induced skin carcinogenesis, concomitantly with decreased OIS. ABCC3 participates in resistance to chemotherapy via its transporter activity. Our data show that this transporter activity is involved in ABCC3-induced senescence, demonstrating that this protein has a complex role in cancer, since its loss of function may promote escape from OIS and tumor formation whereas its gain of function promotes resistance to chemotherapy.

Mdm2 and aurora kinase a inhibitors synergize to block melanoma growth by driving apoptosis and immune clearance of tumor cells.

Therapeutics that induce cancer cell senescence can block cell proliferation and promote immune rejection. However, the risk of tumor relapse due to senescence escape may remain high due to the long lifespan of senescent cells that are not cleared. Here, we show how combining a senescence-inducing inhibitor of the mitotic kinase Aurora A (AURKA) with an MDM2 antagonist activates p53 in senescent tumors harboring wild-type 53. In the model studied, this effect is accompanied by proliferation arrest, mitochondrial depolarization, apoptosis, and immune clearance of cancer cells by antitumor leukocytes in a manner reliant upon Ccl5, Ccl1, and Cxcl9. The AURKA/MDM2 combination therapy shows adequate bioavailability and low toxicity to the host. Moreover, the prominent response of patient-derived melanoma tumors to coadministered MDM2 and AURKA inhibitors offers a sound rationale for clinical evaluation. Taken together, our work provides a preclinical proof of concept for a combination treatment that leverages both senescence and immune surveillance to therapeutic ends.

Irinotecan treatment and senescence failure promote the emergence of more transformed and invasive cells that depend on anti-apoptotic Mcl-1.

Induction of senescence by chemotherapy was initially characterized as a suppressive response that prevents tumor cell proliferation. However, in response to treatment, it is not really known how cells can survive senescence and how irreversible this pathway is. In this study, we analyzed cell escape in response to irinotecan, a first line treatment used in colorectal cancer that induced senescence. We detected subpopulations of cells that adapted to chemotherapy and resumed proliferation. Survival led to the emergence of more transformed cells that induced tumor formation in mice and grew in low adhesion conditions. A significant amount of viable polyploid cells was also generated following irinotecan failure. Markers such as lgr5, CD44, CD133 and ALDH were downregulated in persistent clones, indicating that survival was not associated with an increase in cancer initiating cells. Importantly, malignant cells which resisted senescence relied on survival pathways induced by Mcl-1 signaling and to a lesser extent by Bcl-xL. Depletion of Mcl-1 increased irinotecan efficiency, induced the death of polyploid cells, prevented cell emergence and inhibited growth in low-adhesion conditions. We therefore propose that Mcl-1 targeting should be considered in the future to reduce senescence escape and to improve the treatment of irinotecan-refractory colorectal cancers.

P17 - LYSYL OXIDASES: EMERGING PROMOTERS OF SENESCENCE ESCAPE, TUMOR INITIATION AND PROGRESSION

Lysyl oxidase activity, exhibited by five lox members, has been extensively associated with tumor progression and metastasis. Notably, it is well documented that lox factors, mainly LOX and LOXL2 proteins, are implicated in invasion, cell migration, and angiogenesis in response to tumor-associated hypoxia. In the clinic, their expression correlates with bad prognosis for cancer progression and patient survival. Recently, lox factors have been observed to promote senescence escape, a critical event in tumorigenesis. In two mouse cancer susceptibility models for breast and pancreatic cancer, LOX and/or LOXL2 were found to promote tumorigenesis in cooperation with an oncogenic signal. Mechanistically, lox factors might mediate their effects via several mechanisms; from reorganizing the extracellular matrix and tumor microenvironment, promoting activation of specific pathways such as FAK and Akt pathways, to regulating gene expression by modifying the histone tails on specific target genes such as E-cadherin. This modification impacts the epithelial-mesenchymal transition, a process involved in senescence escape, tumor initiation and progression. The recent literature highlights an important role of lox factors in cancer, and an antibody directed against LOXL2 is already undergoing clinical trials. A better understanding of the biology of these factors will help the design and application of new lox-derived therapeutic tools.

Abstract 1144: Cell plasticity mediated by EMT-inducing transcription factors contributes to melanoma development

Abstract Background: Embryonic transcription factors inducers of Epithelial to Mesenchymal Transition (EMT-TFs) are frequently reactivated during tumorigenesis. In addition to promoting metastasis in carcinoma, they favor neoplastic transformation of epithelial cells by enabling escape from oncogene-induced senescence and apoptosis and providing cells with stem-like properties. We recently unveiled different regulation and function of EMT-TFs in melanoma, a highly metastatic neural crest-derived cancer. We observed a switch in expression from SNAIL2 and ZEB2, which are expressed in melanocytes and behave as oncosuppressive proteins to TWIST1 and ZEB1, which are aberrantly reactivated in melanoma and cooperate with BRAFV600E oncogene to induce neoplastic transformation of melanocytes. This switch in EMT-TF expression represents a novel independent factor of poor prognosis in patients with malignant melanoma (Caramel et al, Cancer Cell, 2013). We further investigated the oncogenic properties of TWIST1 and ZEB1 in melanoma development and analyzed the crosstalk with the master regulator of melanoma phenotypic plasticity, the microphthalmia-associated transcription factor MITF. Methods: We first analysed the expression of MITF and TWIST1/ZEB1 in melanoma cell lines and human melanoma specimens by immunohistochemistry. We then studied the role of TWIST1 in melanoma progression in vivo by crossing conditional Twist1 transgenic mice with BRAFV600E/TyrCreERT2 mice. Results: Our data suggest that EMT-TFs control melanoma cell plasticity by modulating MITF level and the associated pathways. High MITF levels maintain the differentiated status of melanocytes, while reduction of MITF expression results in the transition to proliferative and invasive/stem states. TWIST1 and ZEB1 cooperate with BRAF in downregulating MITF concomitantly with the induction of invasion-associated gene signatures. A correlation of MITF expression with TWIST1 and ZEB1 is observed in human melanoma specimens at the intratumoral level. Moreover, while Twist1 enables BRAFV600E-induced senescence escape in primary mouse melanocytes, monitoring of the development of nevi and melanomas in the BRAFV600E mouse model showed more aggressive features in BRAFV600E/Twist1 mice compared to BRAFV600E control mice. Conclusion: Collectively, these data highlight crucial roles for TWIST1 and ZEB1 in promoting cell dedifferentiation and plasticity of non-epithelial cells. By regulating MITF-dependent phenotype switching they contribute to malignant progression of melanoma. Therefore, targeting the EMT-TF network represents an attractive strategy for metastatic melanomas that invariably develop resistance to BRAFV600 targeted therapy. Citation Format: Julie Caramel, Geoffrey Richard, Michelle Houang, Arnaud de la Fouchardiere, Lionel Larue, Richard Marais, Stephane Dalle, Eugene Tulchinsky, Stephane Ansieau, Alain Puisieux. Cell plasticity mediated by EMT-inducing transcription factors contributes to melanoma development. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1144. doi:10.1158/1538-7445.AM2014-1144

Lysyl oxidases: emerging promoters of senescence escape, tumor initiation and progression

Lysyl oxidases: emerging promoters of senescence escape, tumor initiation and progression

A shorter telomere is the key factor in preventing cultured human mesenchymal stem cells from senescence escape

Mesenchymal stem cells (MSCs) from various animals undergo spontaneous transformation in vitro,establishing some malignant characteristics. However,this phenomenon seems seldom appearing in human (h)MSCs. To address the question whether the hMSCs really do not undergo the spontaneous transformation and why,the present study compared MSCs from two species under the same conditions, the commercialized primary hMSCs whose in vitro life span is very uniform, and the rat (r)MSCs whose spontaneous transformation in vitro is well defined.It was demonstrated that in rMSCs, there were small numbers of re-proliferating cells appearing after a substantial senescent period. These “senescence-escaped”rMSCs were highly proliferative and did not show any sign of growth arrest during the following subcultures upto observed passage 32. Whereas after entering senescence, hMSCs no longer re-proliferated and finally died from apoptosis. Compared with rMSCs, the hMSCs possessed a much shorter telomere, and lacked both telomerase reverse transcriptase expression and telomerase activity. When proliferating from pre-senescent to senescent stages,the hMSCs had a greater loss of relative telomere length(51 % in hMSC vs. 15 % in rMSC), but both cells displayed a similar average telomere shortening per population doubling (0.50 ± 0.06 kb in rMSC vs. 0.49 ± 0.06 kbin hMSC; p > 0.05), indicating that the greater relative shortening of the hMSC telomeres was due to their original shorter length, rather than lack of telomere maintenance mechanisms. In conclusion, the hMSCs do not spontaneously initiate transformation, because they cannot escape senescence. This is particularly due to their much shorter telomere.

Lysyl oxidase activity regulates oncogenic stress response and tumorigenesis

Cellular senescence, a stable proliferation arrest, is induced in response to various stresses. Oncogenic stress-induced senescence (OIS) results in blocked proliferation and constitutes a fail-safe program counteracting tumorigenesis. The events that enable a tumor in a benign senescent state to escape from OIS and become malignant are largely unknown. We show that lysyl oxidase activity contributes to the decision to maintain senescence. Indeed, in human epithelial cell the constitutive expression of the LOX or LOXL2 protein favored OIS escape, whereas inhibition of lysyl oxidase activity was found to stabilize OIS. The relevance of these in vitro observations is supported by in vivo findings: in a transgenic mouse model of aggressive pancreatic ductal adenocarcinoma (PDAC), increasing lysyl oxidase activity accelerates senescence escape, whereas inhibition of lysyl oxidase activity was found to stabilize senescence, delay tumorigenesis, and increase survival. Mechanistically, we show that lysyl oxidase activity favors the escape of senescence by regulating the focal-adhesion kinase. Altogether, our results demonstrate that lysyl oxidase activity participates in primary tumor growth by directly impacting the senescence stability.