Filippa Pettersson

Ribavirin Treatment Effects on Breast Cancers Overexpressing eIF4E, a Biomarker with Prognostic Specificity for Luminal B-Type Breast Cancer

Purpose: We have evaluated the eukaryotic translation initiation factor 4E (eIF4E) as a potential biomarker and therapeutic target in breast cancer. eIF4E facilitates nuclear export and translation of specific, growth-stimulatory mRNAs and is frequently overexpressed in cancer. Experimental Design: Breast cancer cells were treated with ribavirin, an inhibitor of eIF4E, and effects on cell proliferation and on known mRNA targets of eIF4E were determined. eIF4E expression was assessed, at the mRNA and protein level, in breast cancer cell lines and in skin biopsies from patients with metastatic disease. Additionally, pooled microarray data from 621 adjuvant untreated, node-negative breast cancers were analyzed for eIF4E expression levels and correlation with distant metastasis–free survival (DMFS), overall and within each intrinsic breast cancer subtype. Results: At clinically relevant concentrations, ribavirin reduced cell proliferation and suppressed clonogenic potential, correlating with reduced mRNA export and protein expression of important eIF4E targets. This effect was suppressed by knockdown of eIF4E. Although eIF4E expression is elevated in all breast cancer cell lines, variability in ribavirin responsiveness was observed, indicating that other factors contribute to an eIF4E-dependent phenotype. Assessment of the prognostic value of high eIF4E mRNA in patient tumors found that significant discrimination between good and poor outcome groups was observed only in luminal B cases, suggesting that a specific molecular profile may predict response to eIF4E-targeted therapy. Conclusions: Inhibition of eIF4E is a potential breast cancer therapeutic strategy that may be especially promising against specific molecular subtypes and in metastatic as well as primary tumors. Clin Cancer Res; 17(9); 2874–84. ©2011 AACR.

Vorinostat Induces Reactive Oxygen Species and DNA Damage in Acute Myeloid Leukemia Cells

Histone deacetylase inhibitors (HDACi) are promising anti-cancer agents, however, their mechanisms of action remain unclear. In acute myeloid leukemia (AML) cells, HDACi have been reported to arrest growth and induce apoptosis. In this study, we elucidate details of the DNA damage induced by the HDACi vorinostat in AML cells. At clinically relevant concentrations, vorinostat induces double-strand breaks and oxidative DNA damage in AML cell lines. Additionally, AML patient blasts treated with vorinostat display increased DNA damage, followed by an increase in caspase-3/7 activity and a reduction in cell viability. Vorinostat-induced DNA damage is followed by a G2-M arrest and eventually apoptosis. We found that pre-treatment with the antioxidant N-acetyl cysteine (NAC) reduces vorinostat-induced DNA double strand breaks, G2-M arrest and apoptosis. These data implicate DNA damage as an important mechanism in vorinostat-induced growth arrest and apoptosis in both AML cell lines and patient-derived blasts. This supports the continued study and development of vorinostat in AMLs that may be sensitive to DNA-damaging agents and as a combination therapy with ionizing radiation and/or other DNA damaging agents.

Enhanced retinoid-induced apoptosis of MDA-MB-231 breast cancer cells by PKC inhibitors involves activation of ERK

Retinoids are vitamin A derivatives, which cause growth inhibition, differentiation and/or apoptosis in various cell types, including some breast cancer cells. In general, estrogen receptor (ER)-positive cells are retinoic acid (RA) sensitive, whereas ER-negative cells are resistant. In this report, we show that ER-negative MDA-MB-231 cells are strongly growth inhibited by retinoids in combination with a PKC inhibitor. While neither RA nor GF109203X (GF) has a significant growth inhibitory effect in these cells, RA+GF potently suppress proliferation. We found that RA+GF induce apoptosis, as shown by an increase in fragmented DNA, Annexin-V-positive cells and caspase-3 activation. Apoptosis was also induced by GF in combination with two synthetic retinoids. Expression of phosphorylated as well as total PKC was decreased by GF and this was potentiated by RA. In addition, treatment with GF caused a strong and sustained activation of ERK1/2 and p38-MAPK, as well as a weaker activation of JNK. Importantly, inhibition of ERK but not p38 or JNK suppressed apoptosis induced by RA+GF, indicating that activation of ERK is specifically required. In support of this novel finding, the ability of other PKC inhibitors to cause apoptosis in combination with RA correlates with ability to cause sustained activation of ERK.

Genetic and Pharmacologic Inhibition of eIF4E Reduces Breast Cancer Cell Migration, Invasion, and Metastasis

The translation initiation factor eIF4E is an oncogene that is commonly overexpressed in primary breast cancers and metastases. In this article, we report that a pharmacologic inhibitor of eIF4E function, ribavirin, safely and potently suppresses breast tumor formation. Ribavirin administration blocked the growth of primary breast tumors in several murine models and reduced the development of lung metastases in an invasive model. Mechanistically, eIF4E silencing or blockade reduced the invasiveness and metastatic capability of breast cancer cells in a manner associated with decreased activity of matrix metalloproteinase (MMP)-3 and MMP-9. Furthermore, eIF4E silencing or ribavirin treatment suppressed features of epithelial-to-mesenchymal transition, a process crucial for metastasis. Our findings offer a preclinical rationale to explore broadening the clinical evaluation of ribavirin, currently being tested in patients with eIF4E-overexpressing leukemia, as a strategy to treat solid tumors such as metastatic breast cancer.

Expression of Leukemia-Associated Fusion Proteins Increases Sensitivity to Histone Deacetylase Inhibitor–Induced DNA Damage and Apoptosis

Histone deacetylase inhibitors (HDI) show activity in a broad range of hematologic and solid malignancies, yet the percentage of patients in any given malignancy who experience a meaningful clinical response remains small. In this study, we sought to investigate HDI efficacy in acute myeloid leukemia (AML) cells expressing leukemia-associated fusion proteins (LAFP). HDIs have been shown to induce apoptosis, in part, through accumulation of DNA damage and inhibition of DNA repair. LAFPs have been correlated with a DNA repair–deficient phenotype, which may make them more sensitive to HDI-induced DNA damage. We found that expression of the LAFPs PLZF-RARα, PML-RARα, and RUNX1-ETO (AML1-ETO) increased sensitivity to DNA damage and apoptosis induced by the HDI vorinostat. The increase in apoptosis correlated with an enhanced downregulation of the prosurvival protein BCL2. Vorinostat also induced expression of the cell-cycle regulators p19INK4D and p21WAF1 and triggered a G2–M cell cycle arrest to a greater extent in LAFP-expressing cells. The combination of LAFP and vorinostat further led to a greater downregulation of several base excision repair (BER) enzymes. These BER genes represent biomarker candidates for response to HDI-induced DNA damage. Notably, repair of vorinostat-induced DNA double-strand breaks was found to be impaired in PLZF-RARα–expressing cells, suggesting a mechanism by which LAFP expression and HDI treatment cooperate to cause an accumulation of damaged DNA. These data support the continued study of HDI-based treatment regimens in LAFP-positive AMLs. Mol Cancer Ther; 12(8); 1591–604. ©2013 AACR.

Rexinoids Modulate Steroid and Xenobiotic Receptor Activity by Increasing Its Protein Turnover in a Calpain-dependent Manner

The steroid and xenobiotic receptor SXR (human pregnane X receptor) is a nuclear receptor that plays a key role in the bodys detoxification response by regulating genes involved in drug metabolism and transport. SXR ligands include a wide range of compounds, which induce transcription of SXR target genes via activation of a heterodimeric transcription factor consisting of SXR and the related nuclear receptor retinoid X receptor (RXR). We investigated the effect of RXR-selective ligands, rexinoids, on SXR/RXR activity. In agreement with previous reports, we found that rexinoids are weak activators of SXR, but we also found that they can antagonize SXR activation by the potent SXR agonist rifampicin. This antagonism included suppression of rifampicin-induced expression of SXR target genes, as well as reduced binding of SXR/RXR to SXR response elements both in vivo and in vitro. Interestingly, two rexinoids, bexarotene (LGD1069/Targretin®) and LG100268, caused a rapid and sustained decrease in the protein levels of both SXR and RXR. The decrease in SXR level was due to an enhanced rate of protein degradation and was dependent on calpain activity, as opposed to rexinoid-induced RXR degradation, which is mediated via the proteasome. Thus, we have demonstrated a novel, rexinoid-modulated mechanism regulating SXR protein stability, which may explain why rexinoids are only weak activators of SXR/RXR-mediated transcription, despite reports that they bind to SXR with high affinity. We suggest that the ability of rexinoids to induce degradation of both SXR and RXR, in combination with competition for binding to SXR, can also explain why rexinoids antagonize the activation of SXR by drugs like rifampicin.

The Role of eIF4E in Response and Acquired Resistance to Vemurafenib in Melanoma

In eukaryotic cells, the rate-limiting component for cap-dependent mRNA translation is the translation initiation factor eIF4E. eIF4E is overexpressed in a variety of human malignancies, but whether it has a role in melanoma remains obscure. We hypothesized that eIF4E promotes melanoma cell proliferation and facilitates the development of acquired resistance to the BRAF inhibitor vemurafenib. We show that eIF4E is overexpressed in a panel of melanoma cell lines, compared with immortalized melanocytes. Knockdown of eIF4E significantly repressed the proliferation of a subset of melanoma cell lines. Moreover, in BRAF(V600E) melanoma cell lines, vemurafenib inhibits 4E-BP1 phosphorylation, thus promoting its binding to eIF4E. Cap-binding and polysome profiling analysis confirmed that vemurafenib stabilizes the eIF4E-4E-BP1 association and blocks mRNA translation, respectively. Conversely, in cells with acquired resistance to vemurafenib, there is an increased dependence on eIF4E for survival; 4E-BP1 is highly phosphorylated and thus eIF4E-4E-BP1 associations are impeded. Moreover, increasing eIF4E activity by silencing 4E-BP1/2 renders vemurafenib-responsive cells more resistant to BRAF inhibition. In conclusion, these data suggest that therapeutically targeting eIF4E may be a viable means of inhibiting melanoma cell proliferation and overcoming vemurafenib resistance.

The 12th international conference on differentiation therapy: targeting the aberrant growth, differentiation and cell death programs of cancer cells

The 12th international conference on differentiation therapy: targeting the aberrant growth, differentiation and cell death programs of cancer cells

Peroxisomes protect lymphoma cells from HDAC inhibitor-mediated apoptosis

Peroxisomes are a critical rheostat of reactive oxygen species (ROS), yet their role in drug sensitivity and resistance remains unexplored. Gene expression analysis of clinical lymphoma samples suggests that peroxisomes are involved in mediating drug resistance to the histone deacetylase inhibitor (HDACi) Vorinostat (Vor), which promotes ROS-mediated apoptosis. Vor augments peroxisome numbers in cultured lymphoma cells, concomitant with increased levels of peroxisomal proteins PEX3, PEX11B, and PMP70. Genetic inhibition of peroxisomes, using PEX3 knockdown, reveals that peroxisomes protect lymphoma cells against Vor-mediated cell death. Conversely, Vor-resistant cells were tolerant to elevated ROS levels and possess upregulated levels of (1) catalase, a peroxisomal antioxidant, and (2) plasmalogens, ether glycerophospholipids that represent peroxisome function and serve as antioxidants. Catalase knockdown induces apoptosis in Vor-resistant cells and potentiates ROS-mediated apoptosis in Vor-sensitive cells. These findings highlight the role of peroxisomes in resistance to therapeutic intervention in cancer, and provide a novel modality to circumvent drug resistance.

Abstract 4707: Inhibition of autophagy overcomes acquired resistance to vorinostat

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Histone deacetylase inhibitors (HDACi) have recently emerged as promising anticancer agents. Two HDACi, vorinostat (Zolinza®) and romidepsin (Istodax®), are currently FDA approved for use in cutaneous T-cell lymphoma and several others have advanced into Phase II and III clinical trials, both as single agents and in combination with cytotoxics, for a variety of malignancies. Encouraging results have been obtained in haematological malignancies. However, HDACi target many pathways and the full mechanism responsible for their anti-neoplastic activity is still far from clear. An understanding of the molecular mechanisms underlying resistance to HDACi may help to elucidate their mechanism of action and may be of relevance in an attempt to design more effective combination strategies. The purpose of this study is to understand the molecular alterations associated with resistance to vorinostat (Zolinza®). A vorinostat resistant clone (U937-VR) was derived from U937, a monocytic-like histiocytic lymphoma cell line, using a dose escalation protocol. Vorinostat-resistant cells (U937-VR) are able to grow in 2 µM vorinostat without induction of apoptosis. U937-VR cells are cross-resistant to the cytotoxic effects of similar HDACis such as LBH589, but not to the structurally different benzamide MGCD0103, as measured by propidium iodide (PI) staining and caspase 3/7 activation. Also, resistance demonstrates a partial reversibility that could be indicative of a non-mutational mechanism of resistance. The LD50 of different chemotherapeutic drugs was evaluated by measuring apoptosis with PI staining. U937 parental and U937-VR cells have equivalent LD50 for the DNA damaging agent cisplatin and for the microtubule stabilizing agent taxol, indicating that the apoptotic machinery is intact in U937-VR cells. Interestingly, the resistant cells exhibit increased sensitivity toward chloroquine, an inhibitor of autophagy and to the proteasome inhibitors MG132 and bortezomib. This increased sensitivity correlates with an elevated accumulation of ubiquitinated proteins in U937-VR. These cells also have increased autophagic flux which can be inhibited by knock down of Beclin-1 or Lamp-2, which ultimately restores sensitivity to vorinostat. Autophagy is also activated in parental U937 cells upon treatment with vorinostat, however, in contrast to the resistant cells, its inhibition decreases sensitivity to vorinostat. We therefore propose that autophagy switches from a proapoptotic to a prosurvival signal through chronic exposure to vorinostat. The key players involved in that switch remain to be defined. These data are of importance in the design of combination strategies using inhibitors of autophagy and HDACi for the treatment of hematological malignancies. 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 4707. doi:1538-7445.AM2012-4707