Targeting Triple-Negative Breast Cancer With Panobinostat
Introduction Of the more than one million global cases of breast cancer diagnosed each year, approximately fifteen percent are characterized as triple-negative, lacking the estrogen, progesterone, and Her2/neu receptors. Lack of effective therapies, younger age at onset, and early metastatic spread have contributed to the poor prognoses and outcomes associated with these malignancies. Here, we investigate the ability of the histone deacetylase inhibitor panobinostat (LBH589) to selectively target triple-negative breast cancer (TNBC) cell proliferation and survival in vitro and tumorigenesis in vivo.
Methods TNBC cell lines MDA-MB-157, MDA-MB-231, MDA-MB-468, and BT-549 were treated with nanomolar (nM) quantities of panobinostat. Relevant histone acetylation was verified by flow cytometry and immunofluorescent imaging. Assays for trypan blue viability, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation, and DNA fragmentation were used to evaluate overall cellular toxicity. Changes in cell cycle progression were assessed with propidium iodide flow cytometry. Additionally, qPCR arrays were used to probe MDA-MB-231 cells for panobinostat-induced changes in cancer biomarkers and signaling pathways. Orthotopic MDA-MB-231 and BT-549 mouse xenograft models were used to assess the effects of panobinostat on tumorigenesis. Lastly, flow cytometry, ELISA, and immunohistochemical staining were applied to detect changes in cadherin-1, E-cadherin (CDH1) protein expression and the results paired with confocal microscopy in order to examine changes in cell morphology.
Results Panobinostat treatment increased histone acetylation, decreased cell proliferation and survival, and blocked cell cycle progression at G2/M with a concurrent decrease in S phase in all TNBC cell lines. Treatment also resulted in apoptosis induction at 24 hours in all lines except the MDA-MB-468 cell line. MDA-MB-231 and BT-549 tumor formation was significantly inhibited by panobinostat (10 mg/kg/day) in mice. Additionally, panobinostat up-regulated CDH1 protein in vitro and in vivo and induced cell morphology changes in MDA-MB-231 cells consistent with reversal of the mesenchymal phenotype.
Conclusions This study revealed that panobinostat is overtly toxic to TNBC cells in vitro and decreases tumorigenesis in vivo. Additionally, treatment up-regulated anti-proliferative, tumor suppressor, and epithelial marker genes in MDA-MB-231 cells and initiated a partial reversal of the epithelial-to-mesenchymal transition. Our results demonstrate a potential therapeutic role of panobinostat in targeting aggressive triple-negative breast cancer cell types.
Over 200,000 new cases of invasive breast cancer are diagnosed in the United States each year and approximately 40,000 of the patients diagnosed will die from the disease. Breast cancers are routinely classified by stage, pathology, grade and expression of estrogen receptor (ER), progesterone receptor (PR) or human epidermal growth factor receptor (Her2/neu). Current successful therapies include hormone-based agents that directly target these receptors. Triple-negative breast cancer (TNBC) is a heterogeneous subset of neoplasms that is defined by the absence of these targets. Approximately 15% of globally diagnosed breast cancers are designated as ER-, PR- and Her2/neu-negative. Studies have shown that tumors of this aggressive subtype are of higher histological grade, affect a disproportionate number of young women, and are more likely to recur earlier at distant sites, resulting in poor overall prognoses. To improve outcomes of TNBC, we must unravel its biological pathways and modes of progression and use that knowledge to develop novel targets and therapies.
Histone deacetylase inhibitors (HDACis) have emerged as a promising new class of multifunctional anticancer agents. That promise lies in the ability of HDACis to effect multiple epigenetic changes in aberrant cells. In addition to regulating gene expression and transcription through chromatin remodeling, HDACis can also modulate a variety of cellular functions including growth, differentiation, and survival due, in part, to their ability to enhance acetylation of a wide range of proteins, including transcription factors, molecular chaperones, and structural components. Specifically, HDACis have been linked to several downstream effects in tumor cell lines which include: cell cycle arrest, induction of apoptosis, inhibition of angiogenesis, activation or inactivation of tumor suppressor genes or oncogenes, and decreased invasion and metastases.
Panobinostat (LBH589) is a potent pan-deacetylase inhibitor that can block multiple cancer related pathways and reverse epigenetic events implicated in cancer progression. HDACs can be subdivided into two groups: zinc-dependent (Class I, II, and IV) and zinc-independent (Class III). Panobinostat is a potent inhibitor with activity against Class I, II, and IV HDAC enzymes, suggesting true pan-HDAC activity. In preclinical studies, panobinostat has shown potent inhibitory activity at low nanomolar concentrations across a wide range of hematologic malignancies including lymphoma, multiple myeloma and acute myeloid leukemia. It is also being investigated as a treatment against non-responsive solid tumors as well as tumors of the lung, thyroid, and prostate. It has shown synergy with chemotherapeutics, radiation, demethylators, proteasome inhibitors and other agents. Based on these preclinical findings, panobinostat and other HDACis have undergone a rapid phase of clinical development with many entering clinical trials, both as single agents or in combination with other therapies. To date, panobinostat has demonstrated favorable clinical responses, with limited toxicity. There is a critical need to develop pleiotropic therapies that specifically target the neoplasm as well as the biological pathways and markers of TNBC progression. The purpose of this study was to determine the ability of panobinostat to selectively target the TNBC subtype of breast cancer cells, assessed by its effects on the growth, survival, and tumorigenesis of a representative panel of TNBC cells. We also sought to characterize the effects panobinostat on the regulation of breast cancer genes, related signaling pathways and morphology.
Abstract and Introduction
Abstract
Introduction Of the more than one million global cases of breast cancer diagnosed each year, approximately fifteen percent are characterized as triple-negative, lacking the estrogen, progesterone, and Her2/neu receptors. Lack of effective therapies, younger age at onset, and early metastatic spread have contributed to the poor prognoses and outcomes associated with these malignancies. Here, we investigate the ability of the histone deacetylase inhibitor panobinostat (LBH589) to selectively target triple-negative breast cancer (TNBC) cell proliferation and survival in vitro and tumorigenesis in vivo.
Methods TNBC cell lines MDA-MB-157, MDA-MB-231, MDA-MB-468, and BT-549 were treated with nanomolar (nM) quantities of panobinostat. Relevant histone acetylation was verified by flow cytometry and immunofluorescent imaging. Assays for trypan blue viability, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation, and DNA fragmentation were used to evaluate overall cellular toxicity. Changes in cell cycle progression were assessed with propidium iodide flow cytometry. Additionally, qPCR arrays were used to probe MDA-MB-231 cells for panobinostat-induced changes in cancer biomarkers and signaling pathways. Orthotopic MDA-MB-231 and BT-549 mouse xenograft models were used to assess the effects of panobinostat on tumorigenesis. Lastly, flow cytometry, ELISA, and immunohistochemical staining were applied to detect changes in cadherin-1, E-cadherin (CDH1) protein expression and the results paired with confocal microscopy in order to examine changes in cell morphology.
Results Panobinostat treatment increased histone acetylation, decreased cell proliferation and survival, and blocked cell cycle progression at G2/M with a concurrent decrease in S phase in all TNBC cell lines. Treatment also resulted in apoptosis induction at 24 hours in all lines except the MDA-MB-468 cell line. MDA-MB-231 and BT-549 tumor formation was significantly inhibited by panobinostat (10 mg/kg/day) in mice. Additionally, panobinostat up-regulated CDH1 protein in vitro and in vivo and induced cell morphology changes in MDA-MB-231 cells consistent with reversal of the mesenchymal phenotype.
Conclusions This study revealed that panobinostat is overtly toxic to TNBC cells in vitro and decreases tumorigenesis in vivo. Additionally, treatment up-regulated anti-proliferative, tumor suppressor, and epithelial marker genes in MDA-MB-231 cells and initiated a partial reversal of the epithelial-to-mesenchymal transition. Our results demonstrate a potential therapeutic role of panobinostat in targeting aggressive triple-negative breast cancer cell types.
Introduction
Over 200,000 new cases of invasive breast cancer are diagnosed in the United States each year and approximately 40,000 of the patients diagnosed will die from the disease. Breast cancers are routinely classified by stage, pathology, grade and expression of estrogen receptor (ER), progesterone receptor (PR) or human epidermal growth factor receptor (Her2/neu). Current successful therapies include hormone-based agents that directly target these receptors. Triple-negative breast cancer (TNBC) is a heterogeneous subset of neoplasms that is defined by the absence of these targets. Approximately 15% of globally diagnosed breast cancers are designated as ER-, PR- and Her2/neu-negative. Studies have shown that tumors of this aggressive subtype are of higher histological grade, affect a disproportionate number of young women, and are more likely to recur earlier at distant sites, resulting in poor overall prognoses. To improve outcomes of TNBC, we must unravel its biological pathways and modes of progression and use that knowledge to develop novel targets and therapies.
Histone deacetylase inhibitors (HDACis) have emerged as a promising new class of multifunctional anticancer agents. That promise lies in the ability of HDACis to effect multiple epigenetic changes in aberrant cells. In addition to regulating gene expression and transcription through chromatin remodeling, HDACis can also modulate a variety of cellular functions including growth, differentiation, and survival due, in part, to their ability to enhance acetylation of a wide range of proteins, including transcription factors, molecular chaperones, and structural components. Specifically, HDACis have been linked to several downstream effects in tumor cell lines which include: cell cycle arrest, induction of apoptosis, inhibition of angiogenesis, activation or inactivation of tumor suppressor genes or oncogenes, and decreased invasion and metastases.
Panobinostat (LBH589) is a potent pan-deacetylase inhibitor that can block multiple cancer related pathways and reverse epigenetic events implicated in cancer progression. HDACs can be subdivided into two groups: zinc-dependent (Class I, II, and IV) and zinc-independent (Class III). Panobinostat is a potent inhibitor with activity against Class I, II, and IV HDAC enzymes, suggesting true pan-HDAC activity. In preclinical studies, panobinostat has shown potent inhibitory activity at low nanomolar concentrations across a wide range of hematologic malignancies including lymphoma, multiple myeloma and acute myeloid leukemia. It is also being investigated as a treatment against non-responsive solid tumors as well as tumors of the lung, thyroid, and prostate. It has shown synergy with chemotherapeutics, radiation, demethylators, proteasome inhibitors and other agents. Based on these preclinical findings, panobinostat and other HDACis have undergone a rapid phase of clinical development with many entering clinical trials, both as single agents or in combination with other therapies. To date, panobinostat has demonstrated favorable clinical responses, with limited toxicity. There is a critical need to develop pleiotropic therapies that specifically target the neoplasm as well as the biological pathways and markers of TNBC progression. The purpose of this study was to determine the ability of panobinostat to selectively target the TNBC subtype of breast cancer cells, assessed by its effects on the growth, survival, and tumorigenesis of a representative panel of TNBC cells. We also sought to characterize the effects panobinostat on the regulation of breast cancer genes, related signaling pathways and morphology.