Zhewei Zhang

Prostate-targeted radiosensitization via aptamer-shRNA chimeras in human tumor xenografts

Dose-escalated radiation therapy for localized prostate cancer (PCa) has a clear therapeutic benefit; however, escalated doses may also increase injury to noncancerous tissues. Radiosensitizing agents can improve ionizing radiation (IR) potency, but without targeted delivery, these agents will also sensitize surrounding normal tissues. Here we describe the development of prostate-targeted RNAi agents that selectively sensitized prostate-specific membrane antigen-positive (PSMA-positive) cells to IR. siRNA library screens identified DNA-activated protein kinase, catalytic polypeptide (DNAPK) as an ideal radiosensitization target. DNAPK shRNAs, delivered by PSMA-targeting RNA aptamers, selectively reduced DNAPK in PCa cells, xenografts, and human prostate tissues. Aptamer-targeted DNAPK shRNAs, combined with IR, dramatically and specifically enhanced PSMA-positive tumor response to IR. These findings support aptamer-shRNA chimeras as selective sensitizing agents for the improved treatment of high-risk localized PCa.

DNA Damage Recognition via Activated ATM and p53 Pathway in Nonproliferating Human Prostate Tissue

DNA damage response (DDR) pathways have been extensively studied in cancer cell lines and mouse models, but little is known about how DNA damage is recognized by different cell types in nonmalignant, slowly replicating human tissues. Here, we assess, using ex vivo cultures of human prostate tissue, DDR caused by cytotoxic drugs (camptothecin, doxorubicin, etoposide, and cisplatin) and ionizing radiation (IR) in the context of normal tissue architecture. Using specific markers for basal and luminal epithelial cells, we determine and quantify cell compartment-specific damage recognition. IR, doxorubicin, and etoposide induced the phosphorylation of H2A.X on Ser(139) (γH2AX) and DNA damage foci formation. Surprisingly, luminal epithelial cells lack the prominent γH2AX response after IR when compared with basal cells, although ATM phosphorylation on Ser(1981) and 53BP1 foci were clearly detectable in both cell types. The attenuated γH2AX response seems to result from low levels of total H2A.X in the luminal cells. Marked increase in p53, a downstream target of the activated ATM pathway, was detected only in response to camptothecin and doxorubicin. These findings emphasize the diversity of pathways activated by DNA damage in slowly replicating tissues and reveal an unexpected deviation in the prostate luminal compartment that may be relevant in prostate tumorigenesis. Detailed mapping of tissue and cell type differences in DDR will provide an outlook of relevant responses to therapeutic strategies.

Small molecule BMH-compounds that inhibit RNA polymerase I and cause nucleolar stress.

Activation of the p53 pathway has been considered a therapeutic strategy to target cancers. We have previously identified several p53-activating small molecules in a cell-based screen. Two of the compounds activated p53 by causing DNA damage, but this modality was absent in the other four. We recently showed that one of these, BMH-21, inhibits RNA polymerase I (Pol I) transcription, causes the degradation of Pol I catalytic subunit RPA194, and has potent anticancer activity. We show here that three remaining compounds in this screen, BMH-9, BMH-22, and BMH-23, cause reorganization of nucleolar marker proteins consistent with segregation of the nucleolus, a hallmark of Pol I transcription stress. Further, the compounds destabilize RPA194 in a proteasome-dependent manner and inhibit nascent rRNA synthesis and expression of the 45S rRNA precursor. BMH-9– and BMH-22–mediated nucleolar stress was detected in ex vivo–cultured human prostate tissues indicating good tissue bioactivity. Testing of closely related analogues showed that their activities were chemically constrained. Viability screen for BMH-9, BMH-22, and BMH-23 in the NCI60 cancer cell lines showed potent anticancer activity across many tumor types. Finally, we show that the Pol I transcription stress by BMH-9, BMH-22, and BMH-23 is independent of p53 function. These results highlight the dominant impact of Pol I transcription stress on p53 pathway activation and bring forward chemically novel lead molecules for Pol I inhibition, and, potentially, cancer targeting. Mol Cancer Ther; 13(11); 2537–46. ©2014 AACR.

Differential epithelium DNA damage response to ATM and DNA-PK pathway inhibition in human prostate tissue culture

The ability of cells to respond and repair DNA damage is fundamental for the maintenance of genomic integrity. Ex vivo culturing of surgery-derived human tissues has provided a significant advancement to assess DNA damage response (DDR) in the context of normal cytoarchitecture in a non-proliferating tissue. Here, we assess the dependency of prostate epithelium DDR on ATM and DNA-PKcs, the major kinases responsible for damage detection and repair by nonhomologous end-joining (NHEJ), respectively. DNA damage was caused by ionizing radiation (IR) and cytotoxic drugs, cultured tissues were treated with ATM and DNA-PK inhibitors, and DDR was assessed by phosphorylation of ATM and its targets H2AX and KAP1, a heterochromatin binding protein. Phosphorylation of H2AX and KAP1 was fast, transient and fully dependent on ATM, but these responses were moderate in luminal cells. In contrast, DNA-PKcs was phosphorylated in both luminal and basal cells, suggesting that DNA-PK-dependent repair was also activated in the luminal cells despite the diminished H2AX and KAP1 responses. These results indicate that prostate epithelial cell types have constitutively dissimilar responses to DNA damage. We correlate the altered damage response to the differential chromatin state of the cells. These findings are relevant in understanding how the epithelium senses and responds to DNA damage.

Abstract 5387: Prostate-targeted radiosensitization via aptamer-shRNA chimeras

Dose-escalated radiation therapy for localized prostate cancer (PCa) has a clear therapeutic benefit; however, escalated doses may also increase injury to non-cancerous tissues. Radiation-sensitizing agents can improve ionizing radiation (IR) potency, but without targeted delivery these agents will also sensitize surrounding normal tissues. Here we describe the development and application of prostate-targeted RNA interference agents which selectively sensitize PSMA-positive cells to IR. A high throughput siRNA screen identified DNA-PK, MAD2L2, BRCA2, NBN, RAD23B, and RAD54L as candidate DNA-repair pathway targets for prostate cancer radio-sensitization. When candidate shRNAs were conjugated to the PSMA-targeting RNA aptamer, A10-3, they were selectively delivered into PSMA positive prostate cancer cells in the absence of traditional transfection reagents. The shRNA portion was then processed by dicer to the corresponding siRNA. DNA-PK shRNAs, delivered by PSMA-targeting RNA aptamers, selectively reduced DNA-PK in PCa cells, xenografts and human prostate tissues. The mechanism of DNA-PK knock-down was confirmed to be through RNA interference by 5’-RACE assay. In tumor injection models A10-3-DNA-PK-shRNAs, combined with IR, dramatically and specifically enhanced PSMA-positive tumor response to IR. This treatment extended time to reach quadruple tumor volume by approximately 10 weeks when compared to only one week in tumors treated with radiation and control aptamer-shRNAs. A10-3-DNA-PK-shRNA and radiation had no affect on the PSMA negative PC-3 tumors. These studies support aptamer-shRNAs as selective sensitizing agents which may improve the treatment of high-risk localized PCa. 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 5387. doi:10.1158/1538-7445.AM2011-5387

Abstract P3-01-01: Targeting glutamine metabolism in breast cancer for therapy

Metabolic reprogramming of cancer cells is observed in different types of tumors including breast. Oncogenic signals aid changes in metabolism that provide selective advantage to the cancer cells to meet their energy requirements to accomplish rapid proliferation. The increased dependence on the glycolytic pathway for energy called the “Warburg effect” was reported by Otto Warburg several decades ago. Our recent understanding of cancer metabolism has thrown light on alternative energy sources, especially glutamine and other branched chain amino acids. The role of glutamine in breast cancer cell growth has not yet been studied extensively. In this study we found that a number of breast cancer cell lines, especially those negative for ER, PR, HER2, display a high dependence on glutamine for their survival and growth. Interestingly, most of these glutamine-dependent cell lines express high levels of c-myc protein. Consistent with their growth dependency on glutamine, transaminases responsible for entry of glutamine into the tricarboxylic acid cycle are transcriptionally up regulated under low glutamine conditions. Consequently, growth of these cancer cell lines was found to be specifically inhibited by the transaminase inhibitor, amino oxyacetate (AOA). Moreover, the AOA mediated cytotoxic effect was partially c-myc dependent. Through 1H-NMR studies of AOA-treated cells we show that in addition to glutamine, AOA treatment decreases the aspartate and alanine content in the cells. In line with these findings, exogenous supplementation with aspartate partially rescued the cells from the growth inhibitory effects of AOA. Flow cytometry analysis showed that AOA causes cell cycle arrest in the S phase. AOA also had significant inhibitory effect on in vivo growth of rapidly growing SUM 149 and SUM159 xenografts in immunodeficient mice. When combined with chemotherapeutic agents, doxorubicin and carboplatin, AOA inhibited growth of MDA-MB-231 xenograft tumors more effectively than AOA alone. Lastly we present evidence that the cytotoxic effect of AOA is mediated through activation of the ER stress pathway, combined with depletion of key amino acids and likely, a reduction in the nucleoside pool in the cells. Our preclinical studies, both in vitro and in vivo, combined with development of intermediate markers of response, provide a strong rationale for testing AOA for therapy in Phase 0 clinical trials. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-01-01.

Abstract A52: DNA damage responses in different cell types of nonproliferating human prostate tissue

DNA damage response (DDR) pathways have been extensively studied in cancer cell lines and mouse models, but little is known about how DNA damage is recognized by different cell types in nonmalignant, slowly replicating human tissues. Here, we assess, using ex vivo cultures of human prostate tissue, DDR caused by cytotoxic drugs (camptothecin, doxorubicin, etoposide, and cisplatin) and ionizing radiation (IR) in the context of normal tissue architecture. Using specific markers for basal and luminal epithelial cells, we determine and quantify cell compartment-specific damage recognition. IR, doxorubicin, and etoposide induced the phosphorylation of H2A.X on Ser(139) (gH2AX) and DNA damage foci formation. Surprisingly, luminal epithelial cells lack the prominent gH2AX response after IR when compared with basal cells, although ATM phosphorylation on Ser(1981) and 53BP1 foci were clearly detectable in both cell types. The attenuated gH2AX response seems to result from low levels of total H2A.X in the luminal cells. Marked increase in p53, a downstream target of the activated ATM pathway, was detected only in response to camptothecin and doxorubicin. These findings emphasize the diversity of pathways activated by DNA damage in slowly replicating tissues and reveal an unexpected deviation in the prostate luminal compartment that may be relevant in prostate tumorigenesis. Detailed mapping of tissue and cell type differences in DDR will provide an outlook of relevant responses to therapeutic strategies. TSC model presented can also be used for functional genomic studies, cellular and tumor biology as well as for drug response testing. Citation Format: Taija M. af Hallstrom, Sari Jaamaa, Anna Sankila, Tuomas Mirtti, Ville Rantanen, Hannu Koistinen, Ulf-Hakan Stenman, Zhewei Zhang, Zhiming Yang, Angelo M. De Marzo, Kimmo Taari, Mirja Ruutu, Leif C. Andersson, Marikki Laiho. DNA damage responses in different cell types of nonproliferating human prostate tissue. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 626. doi:10.1158/1538-7445.AM2013-626