Escape from p21-mediated oncogene-induced senescence leads to cell dedifferentiation and dependence on anti-apoptotic Bcl-xL and MCL1 proteins.

Oncogene-induced senescence (OIS) is a tumor suppressor response that induces permanent cell cycle arrest in response to oncogenic signaling. Through the combined activation of the p53-p21 and p16-Rb suppressor pathways, OIS leads to the transcriptional repression of proliferative genes. Although this protective mechanism has been essentially described in primary cells, we surprisingly observed in this study that the OIS program is conserved in established colorectal cell lines. In response to the RAS oncogene and despite the inactivation of p53 and p16(INK4), HT29 cells enter senescence, up-regulate p21(WAF1), and induce senescence-associated heterochromatin foci formation. The same effect was observed in response to B-RAF(v600E) in LS174T cells. We also observed that p21(WAF1) prevents the expression of the CDC25A and PLK1 genes to induce cell cycle arrest. Using ChIP and luciferase experiments, we have observed that p21(WAF1) binds to the PLK1 promoter to induce its down-regulation during OIS induction. Following 4-5 weeks, several clones were able to resume proliferation and escape this tumor suppressor pathway. Tumor progression was associated with p21(WAF1) down-regulation and CDC25A and PLK1 reexpression. In addition, OIS and p21(WAF1) escape was associated with an increase in DNA damage, an induction of the epithelial-mesenchymal transition program, and an increase in the proportion of cells expressing the CD24(low)/CD44(high) phenotype. Results also indicate that malignant cells having escaped OIS rely on survival pathways induced by Bcl-xL/MCL1 signaling. In light of these observations, it appears that the transcriptional functions of p21(WAF1) are active during OIS and that the inactivation of this protein is associated with cell dedifferentiation and enhanced survival.

The cdk5 kinase regulates the STAT3 transcription factor to prevent DNA damage upon topoisomerase I inhibition.

The STAT3 transcription factors are cytoplasmic proteins that induce gene activation in response to growth factor stimulation. Following tyrosine phosphorylation, STAT3 proteins dimerize, translocate to the nucleus, and activate specific target genes involved in cell-cycle progression. Despite its importance in cancer cells, the molecular mechanisms by which this protein is regulated in response to DNA damage remain to be characterized. In this study, we show that STAT3 is activated in response to topoisomerase I inhibition. Following treatment, STAT3 is phosphorylated on its C-terminal serine 727 residue but not on its tyrosine 705 site. We also show that topoisomerase I inhibition induced the up-regulation of the cdk5 kinase, a protein initially described in neuronal stress responses. In co-immunoprecipitations, cdk5 was found to associate with STAT3, and pulldown experiments indicated that it associates with the C-terminal activation domain of STAT3 upon DNA damage. Importantly, the cdk5-STAT3 pathway reduced DNA damage in response to topoisomerase I inhibition through the up-regulation of Eme1, an endonuclease involved in DNA repair. ChIP experiments indicated that STAT3 can be found associated with the Eme1 promoter when phosphorylated only on its serine 727 residue and not on tyrosine 705. We therefore propose that the cdk5-STAT3 oncogenic pathway plays an important role in the expression of DNA repair genes and that these proteins could be used as predictive markers of tumors that will fail to respond to chemotherapy.

BCL-XL directly modulates RAS signalling to favour cancer cell stemness.

In tumours, accumulation of chemoresistant cells that express high levels of anti-apoptotic proteins such as BCL-XL is thought to result from the counter selection of sensitive, low expresser clones during progression and/or initial treatment. We herein show that BCL-XL expression is selectively advantageous to cancer cell populations even in the absence of pro-apoptotic pressure. In transformed human mammary epithelial cells BCL-XL favours full activation of signalling downstream of constitutively active RAS with which it interacts in a BH4-dependent manner. Comparative proteomic analysis and functional assays indicate that this is critical for RAS-induced expression of stemness regulators and maintenance of a cancer initiating cell (CIC) phenotype. Resistant cancer cells thus arise from a positive selection driven by BCL-XL modulation of RAS-induced self-renewal, and during which apoptotic resistance is not necessarily the directly selected trait.

Akt inhibition improves irinotecan treatment and prevents cell emergence by switching the senescence response to apoptosis.

Activated in response to chemotherapy, senescence is a tumor suppressive mechanism that induces a permanent loss of proliferation. However, in response to treatment, it is not really known how cells can escape senescence and how irreversible or incomplete this pathway is. We have recently described that cells that escape senescence are more transformed than non-treated parental cells, they resist anoikis and rely on Mcl-1. In this study, we further characterize this emergence in response to irinotecan, a first line treatment used in colorectal cancer. Our results indicate that Akt was activated as a feedback pathway during the early step of senescence. The inhibition of the kinase prevented cell emergence and improved treatment efficacy, both in vitro and in vivo. This improvement was correlated with senescence inhibition, p21waf1 downregulation and a concomitant activation of apoptosis due to Noxa upregulation and Mcl-1 inactivation. The inactivation of Noxa prevented apoptosis and increased the number of emergent cells. Using either RNA interference or p21waf1-deficient cells, we further confirmed that an intact p53-p21-senescence pathway favored cell emergence and that its downregulation improved treatment efficacy through apoptosis induction. Therefore, although senescence is an efficient suppressive mechanism, it also generates more aggressive cells as a consequence of apoptosis inhibition. We therefore propose that senescence-inducing therapies should be used sequentially with drugs favoring cell death such as Akt inhibitors. This should reduce cell emergence and tumor relapse through a combined induction of senescence and apoptosis.

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.

Inhibition of Stat3 by peptide aptamer rS3-PA enhances growth suppressive effects of irinotecan on colorectal cancer cells.

Abstract Cytotoxic agents, alone or in combination, are being used in the treatment of colorectal cancer. Despite progress in the therapeutic regimes, this common malignancy is still the cause of considerable morbidity and mortality, and further improvements are required. Cancer cells often exhibit intrinsic resistance against chemotherapeutic agents or they develop resistance over the time of treatment. Several mechanisms have been made responsible, e.g., drugs may fail to reach tumor cells or drugs may fail to elicit cytotoxicity. The molecular characterization of drug resistance in cancer cells may lead to strategies to overcome it and enhance the sensitivity to chemotherapy. Irinotecan is one of the main treatments of colorectal cancer; it is converted into its active metabolite SN38 and acts as a topoisomerase I inhibitor. Inhibition of this enzyme prevents DNA relegation following uncoiling. Irinotecan has been used as a chemotherapeutic agent either as a single agent or in combination with 5-fluorouracil and targeted therapies directed against the epidermal growth factor receptor, such as cetuximab. The transcription factor signal transducer and activator of transcription 3 (Stat3) is a member of the signal transducer and activator of transcription protein family. Its persistent activation is found in tumor cells and has been associated with drug and radiation resistance. The treatment of colorectal cancer cells with irinotecan leads to senescence or apoptosis following DNA double-strand break induction. This process is impaired by the activation of Stat3. We have derived a Stat3 specific peptide aptamer [recombinant Stat3 inhibitory peptide aptamer (rS3-PA)] that recognizes the dimerization domain of Stat3 and effectively inhibits its function. The delivery of rS3-PA into colon cancer cells and the resulting inhibition of Stat3 strongly enhanced the cytotoxic action of SN38. These data show that the targeted inhibition of Stat3 decreases drug resistance and enhances SN38-mediated cell death. The combination of these agents has a potent antitumor effect and could become beneficial for the treatment of patients with colorectal cancer.