Prasanthi Karna

Cross-talk between Epidermal Growth Factor Receptor and Hypoxia-inducible Factor-1α Signal Pathways Increases Resistance to Apoptosis by Up-regulating Survivin Gene Expression

Although increasing evidence supports a link between epidermal growth factor receptor (EGFR) signaling and resistance to apoptosis, the mechanism by which the EGFR signaling pathway inhibits apoptosis is not well understood. In this study, we found that epidermal growth factor (EGF) stimulation increased the level of expression of the inhibitor of apoptosis protein survivin in breast cancer cells but not in normal mammary epithelial cells. We further demonstrated that activation of survivin gene expression is mediated by oxygen-independent hypoxia-inducible factor (HIF)-1α up-regulation in EGF-treated cancer cells. EGFR signaling activated the phosphoinositide 3-kinase/AKT pathway, subsequently increasing the level of HIF-1α under normoxic conditions. HIF-1α then activated survivin gene transcription through direct binding to the survivin promoter. Furthermore, we found that overexpression of HIF-1α small interfering RNA blocks EGF-induced survivin gene up-regulation and increases apoptosis induced by the chemotherapy drug docetaxel. However, transfection of a plasmid expressing HIF-1α gene activates survivin gene expression and reduces the apoptotic response. Our results demonstrate a novel pathway for EGFR signaling-mediated apoptosis resistance in human cancer cells. Although the role of HIF-1α in regulating cell survival under hypoxic conditions has been studied extensively, our results show that normoxic breast cancer cells utilize cross-talk between EGFR signals and HIF-1α to up-regulate the anti-apoptotic survivin gene, providing a strong rationale for the targeting of HIF-1α as a therapeutic approach for both hypoxic and normoxic tumor cells. Understanding key molecular events in EGFR signaling-induced apoptosis resistance should provide new information for the development of novel therapeutic agents targeting EGFR, HIF-1α, and/or survivin.

Cross-talk between epidermal growth factor receptor and HIF-1 signal pathways increases resistance to apoptosis by upregulating survivin gene expression

Although increasing evidence supports a link between epidermal growth factor receptor (EGFR) signaling and resistance to apoptosis, the mechanism by which the EGFR signaling pathway inhibits apoptosis is not well understood. In this study, we found that epidermal growth factor (EGF) stimulation increased the level of expression of the inhibitor of apoptosis protein survivin in breast cancer cells but not in normal mammary epithelial cells. We further demonstrated that activation of survivin gene expression is mediated by oxygen-independent hypoxia-inducible factor (HIF)-1α up-regulation in EGF-treated cancer cells. EGFR signaling activated the phosphoinositide 3-kinase/AKT pathway, subsequently increasing the level of HIF-1α under normoxic conditions. HIF-1α then activated survivin gene transcription through direct binding to the survivin promoter. Furthermore, we found that overexpression of HIF-1α small interfering RNA blocks EGF-induced survivin gene up-regulation and increases apoptosis induced by the chemotherapy drug docetaxel. However, transfection of a plasmid expressing HIF-1α gene activates survivin gene expression and reduces the apoptotic response. Our results demonstrate a novel pathway for EGFR signaling-mediated apoptosis resistance in human cancer cells. Although the role of HIF-1α in regulating cell survival under hypoxic conditions has been studied extensively, our results show that normoxic breast cancer cells utilize cross-talk between EGFR signals and HIF-1α to up-regulate the anti-apoptotic survivin gene, providing a strong rationale for the targeting of HIF-1α as a therapeutic approach for both hypoxic and normoxic tumor cells. Understanding key molecular events in EGFR signaling-induced apoptosis resistance should provide new information for the development of novel therapeutic agents targeting EGFR, HIF-1α, and/or survivin.

Receptor-Targeted Nanoparticles for In vivo Imaging of Breast Cancer

Purpose: Cell-surface receptor-targeted magnetic iron oxide nanoparticles provide molecular magnetic resonance imaging contrast agents for improving specificity of the detection of human cancer. Experimental Design: The present study reports the development of a novel targeted iron oxide nanoparticle using a recombinant peptide containing the amino-terminal fragment of urokinase-type plasminogen activator (uPA) conjugated to magnetic iron oxide nanoparticles amino-terminal fragment conjugated-iron oxide (ATF-IO). This nanoparticle targets uPA receptor, which is overexpressed in breast cancer tissues. Results: ATF-IO nanoparticles are able to specifically bind to and be internalized by uPA receptor–expressing tumor cells. Systemic delivery of ATF-IO nanoparticles into mice bearing s.c. and i.p. mammary tumors leads to the accumulation of the particles in tumors, generating a strong magnetic resonance imaging contrast detectable by a clinical magnetic resonance imaging scanner at a field strength of 3 tesla. Target specificity of ATF-IO nanoparticles showed by in vivo magnetic resonance imaging is further confirmed by near-IR fluorescence imaging of the mammary tumors using near-IR dye-labeled amino-terminal fragment peptides conjugated to iron oxide nanoparticles. Furthermore, mice administered ATF-IO nanoparticles exhibit lower uptake of the particles in the liver and spleen compared with those receiving nontargeted iron oxide nanoparticles. Conclusions: Our results suggest that uPA receptor–targeted ATF-IO nanoparticles have potential as molecularly targeted, dual modality imaging agents for in vivo imaging of breast cancer.

Down-Regulation of Inhibitor of Apoptosis Proteins by Deguelin Selectively Induces Apoptosis in Breast Cancer Cells

The identification of differentially regulated apoptotic signals in normal and tumor cells allows the development of cancer cell-selective therapies. Increasing evidence shows that the inhibitor of apoptosis (IAP) proteins survivin and XIAP are highly expressed in tumor cells but are absent or have very low levels of expression in normal adult tissues. We found that inhibiting AKT activity with 10 to 100 nM deguelin, a small molecule derived from natural products, markedly reduced the levels of both survivin and XIAP, inducing apoptosis in human breast cancer cells but not in normal cells. It is noteworthy that we detected an elevated level of cleaved poly(ADP-ribose) polymerase, a signature of caspase activation, without a significant increase in caspase activity in deguelin-treated cancer cells. Our results suggest that severe down-regulation of the IAPs by deguelin releases their inhibitory activity over pre-existing active caspases present in cancer cells, inducing apoptosis without the need for further caspase activation. Because normal cells have very low levels of p-AKT, XIAP, survivin, and pre-existing caspase activity, deguelin had little effect on those cells. In addition, we found that combining deguelin with chemotherapy drugs enhanced drug-induced apoptosis selectively in human tumor cells, which suggests that deguelin has great potential for chemosensitization and could represent a new therapeutic agent for treatment of breast cancer.

Regulation of Tat Acetylation and Transactivation Activity by the Microtubule-associated Deacetylase HDAC6

Reversible acetylation of Tat is critical for its transactivation activity toward HIV-1 transcription. However, the enzymes involved in the acetylation/deacetylation cycles have not been fully characterized. In this study, by yeast two-hybrid assay, we have discovered the histone deacetylase HDAC6 to be a binding partner of Tat. Our data show that HDAC6 interacts with Tat in the cytoplasm in a microtubule-dependent manner. In addition, HDAC6 deacetylates Tat at Lys-28 and thereby suppresses Tat-mediated transactivation of the HIV-1 promoter. Inactivation of HDAC6 promotes the interaction of Tat with cyclin T1 and leads to an increase in Tat transactivation activity. These findings establish HDAC6 as a Tat deacetylase and support a model in which Lys-28 deacetylation decreases Tat transactivation activity through affecting the ability of Tat to form a ribonucleoprotein complex with cyclin T1 and the transactivation-responsive RNA.

Targeted Delivery of siRNA‐Generating DNA Nanocassettes Using Multifunctional Nanoparticles

Molecular therapy using a small interfering RNA (siRNA) has shown promise in the development of novel therapeutics. Various formulations have been used for in vivo delivery of siRNAs. However, the stability of short double-stranded RNA molecules in the blood and efficiency of siRNA delivery into target organs or tissues following systemic administration have been the major issues that limit applications of siRNA in human patients. In this study, multifunctional siRNA delivery nanoparticles are developed that combine imaging capability of nanoparticles with urokinase plasminogen activator receptor-targeted delivery of siRNA expressing DNA nanocassettes. This theranostic nanoparticle platform consists of a nanoparticle conjugated with targeting ligands and double-stranded DNA nanocassettes containing a U6 promoter and a shRNA gene for in vivo siRNA expression. Targeted delivery and gene silencing efficiency of firefly luciferase siRNA nanogenerators are demonstrated in tumor cells and in animal tumor models. Delivery of survivin siRNA expressing nanocassettes into tumor cells induces apoptotic cell death and sensitizes cells to chemotherapy drugs. The ability of expression of siRNAs from multiple nanocassettes conjugated to a single nanoparticle following receptor-mediated internalization should enhance the therapeutic effect of the siRNA-mediated cancer therapy.

Activation of EGFR signaling in CD44 positive cells promotes aggressive behavior and progression of breast cancer.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #5056 Ductal carcinoma in situ (DCIS) of the breast is a pre-invasive tumor stage with a hallmark of filling the breast ducts with tumor cells. At present, the mechanism of progression of DCIS to invasive breast cancer is poorly defined. Understanding the molecular events controlling this process should allow for the development of biomarkers for personalized medicine. Increasing evidence suggests that breast cancer is initiated from a few transformed cells that are capable of self-renewal, which is the feature of stem cells. The presence of cancer stem cells in the CD44+/CD24- cells is demonstrated in human breast cancer tissues. We found that 3 to 4% of cells in a human breast cancer cell line, MCF-10DCIS.com (MCF-10DCIS), strongly express CD44. Although MCF-10DCIS cells give rise to DCIS lesions at the early stage which progresses to invasive tumors at a later stage after injection into nude mice, injection of 500 to 5000 of the CD44+ DCIS cells produced predominantly invasive tumors in a short time. However, the CD44 - cells did not produce any tumors even at 100 fold higher cell numbers. We have previously shown that EGFR activated HIF-1α upregulates the anti-apoptotic protein survivin. Our current results show that EGFR-activated HIF-1α plays an important role in the aggressive behavior of CD44+ cells and promotes DCIS progression. Several studies demonstrated that HIF-1α also increases the levels of the genes related to invasion and metastasis, such as uPAR and CXCR4. Histological examination of MCF-10DCIS tumor xenografts shows high levels of CD44, EGFR, HIF-1α and survivin are co-localized at the macroinvasive areas and basal layer of DCIS lesions. Furthermore, invaisve tumor cells are heterogeneous with a high percentage of EGFR+, CD44+, and Her-2 - cells and a low percentage of CD44+, EGFR- and Her-2 + cells. Therefore, this orthotopic model provides an excellent tumor model for identifying biomarkers that promote DCIS invasion. Recent studies have shown that breast cancers with a triple negative phenotype (ER, PR- and Her-2-) are very aggressive and significant less numbers of the cancer patients are found in the DCIS stage compared to other breast cancer types. A high percentage of triple negative cancer tissues also expresses EGFR. We found that CD44 and survivin are expressed in 60 to 70% of the triple negative invasive cancer and in 100% of triple negative DCIS tissues. CD44 and EGFR were detected in triple negative DCIS tumor cells located at the luminal, basal and microinvasive DCIS areas, while in the ER+ DCIS type, CD44 and EGFR are found in the tumor cells at the basal layer and microinvasive foci, suggesting the role of CD44 +, EGFR and survivin expressing tumor cells in progression of DCIS to invasive breast cancer. Since MCF-10DCIS cells are negative for ER and PR, this tumor model provides a great opportunity to investigate signal pathways for triple negative breast cancer as well as the development of novel therapeutics for the treatment of this aggressive cancer type. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5056.

Apoptotic Signaling Pathway and Resistance to Apoptosis in Breast Cancer Stem Cells

A major challenge in the treatment of human breast cancer is the development of resistant mechaims to apoptosis in cancer cells that leads to a low senstivity to therapeutic agents. Recent advances in investigation of the cellular origin of breast cancer showed that breast cancers can be derived from a few tumor initiating cells or cancer stem cells. Increasing evidence supports the notion that cancer stem cells are highly aggressive and resistant to conventional therapies, leading to the progression of breast cancer. Therefore, understanding the molecular mechanisms of differential regulation of the apoptitic signaling pathway in normal mammary epithelial cells, breast cancer stem cells, and breast cancer cells representing different stages of the disease should allow for the development of novel therapeutic approaches targeting dysfunctional apoptotic signaling pathways in breast cancer cells and/or cancer stem~cells.