Senescence is a valid tumor suppressive mechanism in cancer. Accelerated cell senescence describes the growth arrested state of cells that have been treated with anti-tumor drugs, such as doxorubicin that induce a DNA damage response. Discodermolide, a microtubule-stabilizing agent, is a potent inducer of accelerated cell senescence. Resistance to discodermolide is mediated via resistance to accelerated cell senescence, and is associated with reduced expression of the mTORC1 substrate, 4E-BP1 and increased expression of p53 [1]. Although the association of p53 with senescence induction is well-characterized, senescence reversion in the presence of high expression of p53 has not been well-documented. Furthermore, studies addressing the role of mTOR signaling in regulating senescence have been limited and recent data implicate a novel, senescence-associated role for 4E-BP1 in crosstalk with the transcription factor p53. This research perspective will address these somewhat contradictory findings and summarize recent research regarding senescence and mTORC1 signaling.

Discodermolide is a microtubule-stabilizing agent that induces accelerated cell senescence. A discodermolide-resistant cell line, AD32, was generated from the human lung cancer cell line A549. We hypothesize that the major resistance mechanism in these cells is escape from accelerated senescence. AD32 cells have decreased levels of 4E-BP1 mRNA and protein, relative to the parental discodermolide-sensitive A549 cells. Lentiviral-mediated re-expression of wild-type 4E-BP1 in AD32 cells increased the proliferation rate and reverted resistance to discodermolide via restoration of discodermolide-induced accelerated senescence. Consistent with this, cell growth and response to discodermolide was confirmed in vivo using tumor xenograft models. Furthermore, reintroduction of a nonphosphorylatable mutant (Thr-37/46 Ala) of 4E-BP1 was able to partially restore sensitivity and enhance proliferation in AD32 cells, suggesting that these effects are independent of phosphorylation by mTORC1. Microarray profiling of AD32-resistant cells versus sensitive A549 cells, and subsequent unbiased gene ontology analysis, identified molecular pathways and functional groupings of differentially expressed mRNAs implicated in overcoming discodermolide-induced senescence. The most statistically significant classes of differentially expressed genes included p53 signaling, G2/M checkpoint regulation, and genes involved in the role of BRCA1 in the DNA damage response. Consistent with this, p53 protein expression was up-regulated and had increased nuclear localization in AD32 cells relative to parental A549 cells. Furthermore, the stability of p53 was enhanced in AD32 cells. Our studies propose a role for 4E-BP1 as a regulator of discodermolide-induced accelerated senescence. drug resistance | senescence reversion | TOR signaling

SUMMARY There are seven distinct β-tubulin isotypes and eight α-tubulin isotypes in mammals that are hypothesized to have tissue- and cell-specific functions. There is an interest in the use of tubulin isotypes as prognostic markers of malignancy. βV-tubulin, like βIII-tubulin, has been implicated in malignant transformation and drug resistance, however little is known about its localization and function. Thus, we generated for the first time, a rabbit polyclonal antibody specific for human βV-tubulin. The antibody did not cross-react with mouse βV-tubulin or other human β-tubulin isotypes and specifically labeled βV-tubulin by immunoblotting, immunofluorescence and immunohistochemistry. Immunohistochemistry of various human normal tissues revealed that βV-tubulin was expressed in endothelial cells, muscle and cells with muscle differentiation, structures with transport and/or secretary function such as renal tubules, pancreatic ducts and bile ducts, and epithelium with secretary function such as prostate. βV-tubulin was also specifically expressed in pancreatic islets and intratubular germ cell neoplasia, where it may have diagnostic utility. In a small number of malignancies, breast, lung and ovarian cancers, βV-tubulin was aberrantly expressed, suggesting that this isoform may be associated with tumorigenesis. Thus, βV-tubulin expression is a potentially promising prognostic marker of malignancy.

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Introduction: We previously described the association of high tumoral IGF2 expression with reduced disease-free survival in epithelial ovarian tumors. The purpose of this study was to determine the effect of IGF2 overexpression on the tumorigenicity of human ovarian cancer cells. Methods: Expression constructs containing the full-length coding sequence of IGF2 or no insert (empty vector; EV) were made. HEY cells were stably transfected. Quantitative real-time PCR (of CDNA made from cell and xenograft RNA) and ELISA (of conditioned media) were performed to measure the IGF2 mRNA and protein expression levels, respectively. The relative IGF2 mRNA expression level was calculated by the 2−δδCt method with internal normalization to the cyclophilin B mRNA level. Tumorigenicity was evaluated in athymic nude mice by subcutaneous injection of 1×106 cells, with or without matrigel, followed by serial monitoring of in vivo tumor growth by digital caliper measurements. The two-tailed t-test was used to evaluate the significance of comparisons. Results: The IGF2 mRNA level was 5604 ±617 fold-change higher in HEY-IGF2 cells relative to HEY-EV cells (P<0.05). The IGF2 secreted protein level was 1852 ng/ml per 100,000 HEY-IGF2 cells, and was not detectable in the conditioned media of HEY-EV cells. After 4 weeks, the mean tumor volume of subcutaneous xenografts following cell injection without matrigel was 181 ±53 mm3 versus 52 ±15 mm3 for HEY-IGF2 and HEY-EV, respectively (P<0.05, N=10 per group); and for cells injected with matrigel, the mean tumor volume was 1608 ±330 mm3 versus 705 ±95 mm3 for HEY-IGF2 and HEY-EV xenografts, respectively (P<0.05, N=6 per group). The tumor growth rate differed significantly between the HEY-IGF2 and HEY-EV xenografts (P<0.01). In the HEY-IGF2 xenografts, overexpression of IGF2 relative to HEY-EV xenografts was maintained. Conclusions: Stable overexpression of IGF2 in HEY ovarian cancer cells and xenografts was achieved, and resulted in an increase in IGF2 protein secretion. IGF2 overexpression enhanced in vivo tumorigenicity of HEY ovarian cells, accompanied by an increased xenograft growth rate. Further evaluation is underway to fully characterize the mechanisms by which IGF2 overexpression promotes tumor growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A48.

Discodermolide is a microtubule stabilizing agent that induces accelerated cell senescence. A discodermolide-resistant cell line, AD32, was generated from the human lung cancer cell line A549. We hypothesize that the major resistance mechanism in these cells is escape from accelerated senescence. AD32 cells have decreased levels of 4E-BP1 mRNA and protein, relative to the parental discodermolide-sensitive A549 cells. Lentiviral-mediated re-expression of wild type 4E-BP1 in AD32 cells increased the proliferation rate and reverted resistance to discodermolide via restoration of discodermolide-induced accelerated senescence. Consistent with this, cell growth and response to discodermolide was confirmed in vivo using tumor xenograft models. Furthermore, re-introduction of a non-phosphorylatable mutant (Thr 37/46 Ala) of 4E-BP1 was able to partially restore sensitivity and enhance proliferation in AD32 cells, suggesting that these effects are independent of phosphorylation by mTORC1. Microarray profiling of AD32 resistant cells versus sensitive A549 cells, and subsequent unbiased gene ontology analysis, identified molecular pathways and functional groupings of differentially expressed mRNAs implicated in overcoming discodermolide-induced senescence. The most-statistically significant classes of differentially expressed genes included p53 signaling, G2/M checkpoint regulation and genes involved in the role of BRCA1 in the DNA damage response. Consistent with this, p53 protein expression was up-regulated and had increased nuclear localization in AD32 cells relative to parental A549 cells. Furthermore, the stability of p53 was enhanced in AD32 cells. Our studies propose a role for 4E-BP1 as a regulator of discodermolide-induced accelerated senescence. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1228. doi:10.1158/1538-7445.AM2011-1228

Introduction High Insulin-like Growth Factor 2 (IGF2) tumor expression correlates with reduced disease-free survival in epithelial ovarian tumors. IGF2 mRNA is also upregulated after Taxol treatment of ovarian cancer cells. The purpose of this study was to determine the effect of IGF2 overexpression on the tumorigenicity of human ovarian cancer cells. Methods HEY cells were stably transfected using lentiviral particles with IGF2 or no insert (EV) constructs. qPCR and ELISA were performed to measure the IGF2 mRNA and secreted protein expression levels, respectively. Signaling pathways were probed by immunoblotting. Tumorigenicity was evaluated in athymic nude mice by subcutaneous injection of serial cell dilutions with matrigel, followed by monitoring of in vivo tumor growth by caliper measurements. qPCR for IGF2 mRNA and immunohistochemistry for Ki67 were performed on the excised tumors. Results The IGF2 mRNA level was 4679 ±176 fold-change higher in HEY-IGF2 cells relative to HEY-EV cells (P<0.0001). The amount of IGF2 protein secreted in 48 h was 5159 ng/100,000 HEY-IGF2 cells (51.6 pg/cell), and undetectable in the media of HEY-EV cells. Tyrosine phosphorylation of the IGF1-receptor was increased 13-fold in HEY-IGF2 cells. In vitro, dependency on the IGF1-receptor signaling was unaltered, as IGF2 overexpressing cells responded similarly to the IGF1-receptor inhibitor NVP-AEW541 compared to HEY-EV cells. Following an injection of one million cells with matrigel, the mean tumor volume of xenografts after 4 weeks was 1608 ±330 mm3 versus 705 ±95 mm3 for HEY-IGF2 and HEY-EV xenografts, respectively (P=0.04, N=6 per group). Immunohistochemistry for Ki67 showed a significantly higher percentage of stained nuclei in HEY-IGF2 tumors than in HEY-EV tumors (P=0.04, N=3 per group). Discussion Stable overexpression of IGF2 in HEY ovarian cancer cells resulted in an increased xenograft growth rate. Further evaluation is underway to fully characterize the mechanisms by which IGF2 overexpression promotes tumor growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1080. doi:1538-7445.AM2012-1080