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Cisplatin resistance occurs, at least in part, through the function of the Fanconi anemia (FA)/BRCA pathway, a DNA-damage response pathway required for repair of cisplatin cross-links. In the current study, we designed a cell-based screening strategy to identify small-molecule inhibitors of the FA/BRCA pathway with the hypothesis that such molecules could restore sensitivity to platinum agents. We identified four inhibitors, including three protein kinase inhibitors (wortmannin, H-9, and alsterpaullone) and one natural compound (curcumin) that inhibit the FA/BRCA pathway. We show that curcumin, a compound that is generally regarded as safe, inhibits the monoubiquitination of the FANCD2 protein as predicted by the screen and consequently sensitizes ovarian and breast tumor cell lines to cisplatin through apoptotic cell death. We believe that this study shows an efficient, high-throughput method for identifying new compounds that may sensitize cancer cells to DNA-damaging chemotherapy. [Mol Cancer Ther 2006;5(4):952–61]

Background: CXCL8 signaling levels are elevated in localized high grade prostate cancer (CaP). We have shown exposure of CaP cells to chemical DNA-damaging agents potentiates CXCL8 signaling, which underpins an autocrine mode of therapeutic resistance. Data from in vitro and in vivo CaP models has shown loss of the tumor suppressor PTEN also increases expression of the pro-inflammatory chemokine CXCL8. Experiments were designed to determine whether exposure to ionising radiation (IR) selectively induced CXC-chemokine signaling in PTEN-deficient CaP cells, and to determine how CXCL8 signaling may modulate their radiosensitivity. Methods: shRNA-directed strategies were used to establish cell-based models of CaP with differential expression of PTEN. These clonal (DU145) and heterogeneous (22Rv1) populations were subjected to IR under normal or hypoxic culture conditions. PTEN was reconstituted in PC3 cells and was driven by tetracycline-regulated expression of the gene. Changes in chemokine expression were analyzed by RT-PCR, immunoblotting and ELISA. Autocrine CXCL8 signaling was attenuated using siRNA to down-regulate gene expression of its receptors, CXCR1 and CXCR2. The effects of modulating chemokine signaling on cell viability following exposure to clinically-relevant doses of IR were assessed by colony formation assays, cell proliferation assays, and analysis of apoptosis and cellular senescence. The effect of CXCL8 signaling inhibition in vivo was determined using a PC3 xenograft model in SCID mice. The cell penetrating pepducin, x1/2pal-i3, was used to attenuate both CXCR1 and CXCR2 and tumor growth was assessed via caliper measurements. Results: Exposure to IR (3 Gy) increased gene expression of CXCL8, CXCR1 and CXCR2 - with PTEN-deficiency enhancing this response. Loss of PTEN expression differentially modulated the radiosensitivty of CaP cell lines: PTEN-depletion conferred relative radioresistance in DU145 cells but made 22RV1 cells more radiosensitive. However, the inhibition of CXCL8 signaling in all PTEN-depleted models resulted in consistently increased radiosensitivity (SER 1.2, 1.13, and 1.39 in DU145, 22RV1, and PC3 CaP cells, respectively; p Conclusions: Our results demonstrate the role of treatment-induced CXCL8 signaling as a mechanism of resistance to IR in PTEN-depleted CaP cells. Inhibition of IR-induced CXCL8 signaling provides a novel therapeutic opportunity to enhance the radiosensitivity of PTEN-depleted CaP cells. However, mechanistic results indicate that other genetic factors including p53 status dictate the resultant induction of treatment-induced apoptosis. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C215. Citation Format: Chris WD Armstrong, Jonathan A. Coulter, Steven Walker, Richard D. Kennedy, Kevin M. Prise, Joe M. O'Sullivan, David JJ Waugh. Sensitivity of PTEN-deficient prostate carcinoma xenografts to ionizing radiation through inhibition of treatment-induced CXCL8 signaling. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C215.

Background: PTEN loss is associated with early prostate cancer progression. We sought to characterize whether PTEN loss regulates pro-inflammatory chemokine signaling and its functional significance to PTEN-depleted prostate cancer cells. Methods: Chemokine/chemokine receptor expression was assessed in vitro in established cell lines, exhibiting different levels of endogenous PTEN expression. Down-regulation of endogenous PTEN expression in DU145 and 22Rv1 cells was effected using transient siRNA or stable shRNA interventions. Chemokine expression was further evaluated by immunohistochemistry in prostate tissue from wild-type and PTEN+/− mice. Results: Loss of PTEN induced transcription-mediated increases in expression of CXCL8 and its receptors, CXCR1 and CXCR1 in DU145 and 22Rv1 cells. Stress-induced expression of CXCL8, CXCR1 and CXCR2 was greater in magnitude and duration in PTEN-null PC3 and LNCaP cells, in contrast to the dampened response observed in PTEN-expressing cells. PTEN loss had no effect on basal or stress-induced expression of CCL2, CXCL12 or their receptors CCR2 or CXCR4 in prostate cancer cells. The efficacy of CXCL8 signaling upon HIF-1 and NF-B transcription was also greater in magnitude and duration in PTEN-null prostate cancer cells or following siRNA-mediated PTEN knockdown. Increased expression of the orthologous murine chemokine KC was elevated in atypical cytological features of PTEN+/− prostate tissue relative to normal epithelium in PTENWT glands. Attenuation of CXCL8 signaling using siRNA oligonucleotides or shRNA vectors in cell-based models of partial or complete PTEN-loss significantly decreased cell viability. Furthermore, attenuation of CXCL8 signaling modulates the response of PTEN-depleted prostate cancer cells to clinically-relevant doses of ionizing radiation. Conclusion: PTEN loss promotes a selective up-regulation of CXCL8 signaling and increases the efficacy of CXCL8 signaling in prostate cancer cells. Functionally, this selective induction of CXCL8 signaling acts to maintain cell viability as an essential adaptation to the genetic instability arising from PTEN loss. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B200.