Yunguang Sun

Targeting the Mechanisms of Resistance to Chemotherapy and Radiotherapy with the Cancer Stem Cell Hypothesis

Despite advances in treatment, cancer remains the 2nd most common cause of death in the United States. Poor cure rates may result from the ability of cancer to recur and spread after initial therapies have seemingly eliminated detectable signs of disease. A growing body of evidence supports a role for cancer stem cells (CSCs) in tumor regrowth and spread after initial treatment. Thus, targeting CSCs in combination with traditional induction therapies may improve treatment outcomes and survival rates. Unfortunately, CSCs tend to be resistant to chemo- and radiation therapy, and a better understanding of the mechanisms underlying CSC resistance to treatment is necessary. This paper provides an update on evidence that supports a fundamental role for CSCs in cancer progression, summarizes potential mechanisms of CSC resistance to treatment, and discusses classes of drugs currently in preclinical or clinical testing that show promise at targeting CSCs.

Inhibition of JAK2 signaling by TG101209 enhances radiotherapy in lung cancer models.

Introduction: Persistent STAT3 activation contributes to lung carcinogenesis. Survivin, one of STAT3-regulated genes, is antiapoptotic and confers cancer radioresistance. Methods: We tested whether TG101209, a small-molecule inhibitor of JAK2 (a STAT3-activating tyrosine kinase), affected survivin expression and sensitized lung cancer to radiation. We investigated whether inhibition of JAK2 signaling with TG101209 can be used to reduce survivin expression and enhance radiosensitivity of lung cancer cells in vitro and tumor growth delay in vivo. JAK2 downstream signaling, including PI3-K/Akt and Ras/MAPK/ERK pathways, was also explored. Results: TG101209 inhibited STAT3 activation and survivin expression and sensitized HCC2429 (dose enhancement ratio = 1.34, p = 0.002) and H460 (dose enhancement ratio = 1.09, p = 0.006) cells to radiation in clonogenic assays. Radiation promoted phospho-Akt and phospho-ERK in H460 cells, while their levels were unchanged in HCC2429. After treatment with TG101209, phospho-ERK protein levels were reduced in both HCC2429 and H460 cells. HCC2429 cells transfected with KRAS-12V mutant were more resistant to radiation- and TG101209-induced apoptosis than wild-type control cells. In vivo, addition of TG101209 to radiation in lung xenografts produced a significant tumor growth delay (>10 days) compared with radiation alone and was well tolerated. Immunohistochemistry staining of tumor sections showed that TG101209 increased apoptosis and decreased cell proliferation and vascular density, suggesting that TG101209 also has antiangiogenic effects. Conclusions: TG101209 enhanced the effects of radiation in lung cancer in vitro and in vivo. This study suggests the potential utility of selecting lung cancer patients according to KRAS mutation status for future clinical trials testing combination of TG101209 and radiotherapy.

Modulation of transcription parameters in glucocorticoid receptor-mediated repression.

Glucocorticoid receptors (GRs) affect both gene induction and gene repression. The disparities of receptor binding to DNA and increased vs. decreased gene expression have suggested significant mechanistic differences between GR-mediated induction and repression. Numerous transcription factors are known to modulate three parameters of gene induction: the total activity (Vmax) and position of the dose-response curve with glucocorticoids (EC50) and the percent partial agonist activity with antiglucocorticoids. We have examined the effects on GR-mediated repression of five modulators (coactivators TIF2 [GRIP1, SRC-2] and SRC-1, corepressor SMRT, and comodulators STAMP and Ubc9), a glucocorticoid steroid (deacylcortivazol [DAC]) of very different structure, and an inhibitor of histone deacetylation (trichostatin A [TSA]). These factors interact with different domains of GR and thus are sensitive topological probes of GR action. These agents altered the Vmax, EC50, and percent partial agonist activity of endogenous and exogenous repressed genes similarly to that previously observed for GR-regulated gene induction. Collectively, these results suggest that GR-mediated induction and repression share many of the same molecular interactions and that the causes for different levels of gene transcription arise from more distal downstream steps.

Role of Insulin-Like Growth Factor-1 Signaling Pathway in Cisplatin-Resistant Lung Cancer Cells

PURPOSE The development of drug-resistant phenotypes has been a major obstacle to cisplatin use in non-small-cell lung cancer. We aimed to identify some of the molecular mechanisms that underlie cisplatin resistance using microarray expression analysis. METHODS AND MATERIALS H460 cells were treated with cisplatin. The differences between cisplatin-resistant lung cancer cells and parental H460 cells were studied using Western blot, MTS, and clonogenic assays, in vivo tumor implantation, and microarray analysis. The cisplatin-R cells were treated with human recombinant insulin-like growth factor (IGF) binding protein-3 and siRNA targeting IGF-1 receptor. RESULTS Cisplatin-R cells illustrated greater expression of the markers CD133 and aldehyde dehydrogenase, more rapid in vivo tumor growth, more resistance to cisplatin- and etoposide-induced apoptosis, and greater survival after treatment with cisplatin or radiation than the parental H460 cells. Also, cisplatin-R demonstrated decreased expression of insulin-like growth factor binding protein-3 and increased activation of IGF-1 receptor signaling compared with parental H460 cells in the presence of IGF-1. Human recombinant IGF binding protein-3 reversed cisplatin resistance in cisplatin-R cells and targeting of IGF-1 receptor using siRNA resulted in sensitization of cisplatin-R-cells to cisplatin and radiation. CONCLUSIONS The IGF-1 signaling pathway contributes to cisplatin-R to cisplatin and radiation. Thus, this pathway represents a potential target for improved lung cancer response to treatment.

Effects of acetylation, polymerase phosphorylation, and DNA unwinding in glucocorticoid receptor transactivation

Varying the concentration of selected factors alters the induction properties of steroid receptors by changing the position of the dose-response curve (or the value for half-maximal induction=EC(50)) and the amount of partial agonist activity of antisteroids. We now describe a rudimentary mathematical model that predicts a simple Michaelis-Menten curve for the multi-step process of steroid-regulated gene induction. This model suggests that steps far downstream from receptor binding to steroid can influence the EC(50) of agonist-complexes and partial agonist activity of antagonist-complexes. We therefore asked whether inhibitors of three possible downstream steps can reverse the effects of increased concentrations of two factors: glucocorticoid receptors (GRs) and Ubc9. The downstream steps (with inhibitors in parentheses) are protein deacetylation (TSA and VPA), DNA unwinding (CPT), and CTD phosphorylation of RNA polymerase II (DRB and H8). None of the inhibitors mimic or prevent the effects of added GRs. However, inhibitors of DNA unwinding and CTD phosphorylation do reverse the effects of Ubc9 with high GR concentrations. These results support our earlier conclusion that different rate-limiting steps operate at low and high GR concentrations versus high GR with Ubc9. The present data also suggest that downstream steps can modulate the EC(50) of GR-mediated induction, thus both supporting the utility of our mathematical model and widening the field of biochemical processes that can modify the EC(50).

ALK Inhibitor PF02341066 (Crizotinib) Increases Sensitivity to Radiation in Non–Small Cell Lung Cancer Expressing EML4-ALK

Crizotinib (PF02341066) is a tyrosine kinase inhibitor of anaplastic lymphoma kinase (ALK) that has been shown to selectively inhibit growth of cancer cells that harbor the EML4-ALK fusion found in a subset of patients with non–small cell lung cancer (NSCLC). While in clinical trials, PF02341066 has shown a significant therapeutic benefit as a single agent; the effectiveness of combining it with other therapeutic modalities including ionizing radiation remains unknown. To further elucidate the role of PF02341066 in tumor inhibition, we examined its effects alone and in combination with radiation on downstream signaling, apoptosis, and radiosensitivity in two NSCLC cell lines in vitro: H3122, which harbors the EML4-ALK fusion, and H460, which does not. We also examined the in vivo effects of PF02341066 in H3122 mouse xenografts. In the H3122 cell line, PF02341066 inhibited phosphorylation of ALK and its downstream effectors: AKT, ERK, and STAT3. H3122 cells treated with a combination of PF02341066 and radiation showed an increase in cellular apoptosis and were sensitized to radiation therapy (dose enhancement ratio, 1.43; P < 0.0001). Moreover, in an H3122 xenograft model, the combined treatment resulted in greater tumor growth inhibition than either treatment alone (P < 0.05). None of these effects was observed in the EML4-ALK–negative H460 cells. Our findings indicate that PF02341066 acts as a radiation sensitizer in cells harboring the EML4-ALK fusion, providing a rationale for a clinical trial combining ALK inhibitor with radiation in the NSCLCs expressing ALK. Mol Cancer Ther; 12(5); 696–704. ©2013 AACR.

Enhanced Radiosensitivity of Androgen-Resistant Prostate Cancer: AZD1152-Mediated Aurora Kinase B Inhibition

Aurora kinase B (AURKB) is critical to the process of mitosis, aiding in chromosome condensation by phosphorylating histone H3. We investigated the effects of AZD1152, an AURKB inhibitor, on radiosensitivity of androgen-insensitive prostate cancer cells. The goal of this study was to test whether AZD1152 increases the susceptibility of hormone-refractory prostate cancer cells to radiation-induced DNA damage and to determine the conditions of AZD1152 treatment that maximize radiosensitization. PC3 and DU145 cells were treated with various AZD1152 doses for various durations to elucidate the conditions that yielded maximal increases in G2/M-phase and polyploid cells. To assess DNA damage, &ggr;-H2AX phosphorylation was quantified for cells grown under radiosensitizing conditions and subjected to either no radiation or 5 Gy radiation. Radiosensitivity was determined by clonogenic assays. Cell cycle effects in both cell lines were maximized by treatment with 60 nM AZD1152 for 48 h. AZD1152-treated cells exhibited significantly increased DNA damage 30 min postirradiation (PC3: 100% compared to 68%, P  =  0.035; DU145: 100% compared to 69%, P  =  0.034), with additional DNA damage 6 h postirradiation (PC3: 85% compared to 15%, P  =  0.002; DU145: 67% compared to 21%, P  =  0.012). Radiosensitivity was increased in both cell lines, with dose enhancement ratios of 1.53 for PC3 cells (P  =  0.017) and 1.71 for DU145 cells (P  =  0.02). This study identifies the optimal AZD1152 treatment conditions to maximize the radiosensitization of PC3 and DU145 cells. These results suggest a major role for DNA damage and impairment of DNA repair mechanisms in AZD1152-induced radiosensitization of prostate cancer cells.

PA1 Protein, a New Competitive Decelerator Acting at More than One Step to Impede Glucocorticoid Receptor-mediated Transactivation

Background: Unexpected PA1 binding to glucocorticoid receptors (GRs) suggests a role in gene transactivation. Results: PA1 suppresses induction properties (total agonist activity, EC50, and partial agonist activity of antagonist) of exogenous and endogenous genes by GRs and other receptors. Conclusion: Two different assays indicate PA1 inhibits GR-regulated gene induction at two distinct steps. Significance: Dual-site action of PA1 may be utilized by other transcriptional cofactors. Numerous cofactors modulate the gene regulatory activity of glucocorticoid receptors (GRs) by affecting one or more of the following three major transcriptional properties: the maximal activity of agonists (Amax), the potency of agonists (EC50), and the partial agonist activity of antisteroids (PAA). Here, we report that the recently described nuclear protein, Pax2 transactivation domain interaction protein (PTIP)-associated protein 1 (PA1), is a new inhibitor of GR transactivation. PA1 suppresses Amax, increases the EC50, and reduces the PAA of an exogenous reporter gene in a manner that is independent of associated PTIP. PA1 is fully active with, and strongly binds to, the C-terminal half of GR. PA1 reverses the effects of the coactivator TIF2 on GR-mediated gene induction but is unable to augment the actions of the corepressor SMRT. Analysis of competition assays between PA1 and TIF2 with an exogenous reporter indicates that the kinetic definition of PA1 action is a competitive decelerator at two sites upstream from where TIF2 acts. With the endogenous genes IGFBP1 and IP6K3, PA1 also represses GR induction, increases the EC50, and decreases the PAA. ChIP and re-ChIP experiments indicate that PA1 accomplishes this inhibition of the two genes via different mechanisms as follows: PA1 appears to increase GR dissociation from and reduce GR transactivation at the IGFBP1 promoter regions but blocks GR binding to the IP6K3 promoter. We conclude that PA1 is a new competitive decelerator of GR transactivation and can act at more than one molecularly defined step in a manner that depends upon the specific gene.

BV6, an IAP Antagonist, Activates Apoptosis and Enhances Radiosensitization of Non-small Cell Lung Carcinoma In Vitro

Introduction: Defects in the apoptosis pathway limit the effectiveness of radiation in non-small cell lung cancer (NSCLC) therapy. BV6 is an antagonist of cIAP1 and XIAP, members of the inhibitors of apoptosis (IAP) family. We investigated the potential of BV6 to sensitize NSCLC cell lines to radiation. Methods: HCC193 and H460 lung cancer cell lines were treated with BV6 to investigate the effects of drug administration on cell proliferation, apoptosis, inhibition of XIAP and cIAP1, and radiosensitivity. Subsequent immunoblotting and Hoechst staining were used to determine the role of apoptosis in radiosensitization. Finally, the pathway of apoptosis was characterized by Western blot analysis for cleaved caspase-8 and cleaved caspase-9 and enzyme-linked immunosorbent assays for TNF-&agr;. Results: HCC193 was found to be more sensitive than H460 to BV6-induced apoptosis in a concentration-dependent and time-dependent manner. BV6 significantly sensitized both cell lines to radiation (HCC193—DER = 1.38, p < 0.05 at 1 &mgr;M BV6; H460—DER = 1.42, p < 0.05 at 5 &mgr;M BV6), but a higher concentration of and longer incubation time with BV6 was necessary for H460 cells. The BV6-induced radiosensitization of HCC193 favored the extrinsic pathway of apoptosis, whereas that of H460 favored the intrinsic pathway. Conclusions: BV6, an IAP antagonist, significantly enhanced the radiosensitization of HCC193 and H460 cells in vitro. More research is warranted to test the mechanism of action of BV6 and to assess its potential in vivo and in the clinical setting.

Higher levels of c-Met expression and phosphorylation identify cell lines with increased sensitivity to AMG-458, a novel selective c-Met inhibitor with radiosensitizing effects.

PURPOSE c-Met is overexpressed in some non-small cell lung cancer (NSCLC) cell lines and tissues. Cell lines with higher levels of c-Met expression and phosphorylation depend on this receptor for survival. We studied the effects of AMG-458 on 2 NSCLC cell lines. METHODS AND MATERIALS 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assays assessed the sensitivities of the cells to AMG-458. Clonogenic survival assays illustrated the radiosensitizing effects of AMG-458. Western blot for cleaved caspase 3 measured apoptosis. Immunoblotting for c-Met, phospho-Met (p-Met), Akt/p-Akt, and Erk/p-Erk was performed to observe downstream signaling. RESULTS AMG-458 enhanced radiosensitivity in H441 but not in A549. H441 showed constitutive phosphorylation of c-Met. A549 expressed low levels of c-Met, which were phosphorylated only in the presence of exogenous hepatocyte growth factor. The combination of radiation therapy and AMG-458 treatment was found to synergistically increase apoptosis in the H441 cell line but not in A549. Radiation therapy, AMG-458, and combination treatment were found to reduce p-Akt and p-Erk levels in H441 but not in A549. H441 became less sensitive to AMG-458 after small interfering RNA knockdown of c-Met; there was no change in A549. After overexpression of c-Met, A549 became more sensitive, while H441 became less sensitive to AMG-458. CONCLUSIONS AMG-458 was more effective in cells that expressed higher levels of c-Met/p-Met, suggesting that higher levels of c-Met and p-Met in NSCLC tissue may classify a subset of tumors that are more sensitive to molecular therapies against this receptor.