Clarissa Amaya

Propranolol treatment of infantile hemangioma endothelial cells: A molecular analysis

Infantile hemangiomas (IHs) are non-malignant, largely cutaneous vascular tumors affecting approximately 5–10% of children to varying degrees. During the first year of life, these tumors are strongly proliferative, reaching an average size ranging from 2 to 20 cm. These lesions subsequently stabilize, undergo a spontaneous slow involution and are fully regressed by 5 to 10 years of age. Systemic treatment of infants with the non-selective β-adrenergic receptor blocker, propranolol, has demonstrated remarkable efficacy in reducing the size and appearance of IHs. However, the mechanism by which this occurs is largely unknown. In this study, we sought to understand the molecular mechanisms underlying the effectiveness of β blocker treatment in IHs. Our data reveal that propranolol treatment of IH endothelial cells, as well as a panel of normal primary endothelial cells, blocks endothelial cell proliferation, migration, and formation of the actin cytoskeleton coincident with alterations in vascular endothelial growth factor receptor-2 (VEGFR-2), p38 and cofilin signaling. Moreover, propranolol induces major alterations in the protein levels of key cyclins and cyclin-dependent kinase inhibitors, and modulates global gene expression patterns with a particular affect on genes involved in lipid/sterol metabolism, cell cycle regulation, angiogenesis and ubiquitination. Interestingly, the effects of propranolol were endothelial cell-type independent, affecting the properties of IH endothelial cells at similar levels to that observed in neonatal dermal microvascular and coronary artery endothelial cells. This data suggests that while propranolol markedly inhibits hemangioma and normal endothelial cell function, its lack of endothelial cell specificity hints that the efficacy of this drug in the treatment of IHs may be more complex than simply blockage of endothelial function as previously believed.

Targeting of Beta Adrenergic Receptors Results in Therapeutic Efficacy against Models of Hemangioendothelioma and Angiosarcoma

Therapeutic targeting of the beta-adrenergic receptors has recently shown remarkable efficacy in the treatment of benign vascular tumors such as infantile hemangiomas. As infantile hemangiomas are reported to express high levels of beta adrenergic receptors, we examined the expression of these receptors on more aggressive vascular tumors such as hemangioendotheliomas and angiosarcomas, revealing beta 1, 2, and 3 receptors were indeed present and therefore aggressive vascular tumors may similarly show increased susceptibility to the inhibitory effects of beta blockade. Using a panel of hemangioendothelioma and angiosarcoma cell lines, we demonstrate that beta adrenergic inhibition blocks cell proliferation and induces apoptosis in a dose dependent manner. Beta blockade is selective for vascular tumor cells over normal endothelial cells and synergistically effective when combined with standard chemotherapeutic or cytotoxic agents. We demonstrate that inhibition of beta adrenergic signaling induces large scale changes in the global gene expression patterns of vascular tumors, including alterations in the expression of established cell cycle and apoptotic regulators. Using in vivo tumor models we demonstrate that beta blockade shows remarkable efficacy as a single agent in reducing the growth of angiosarcoma tumors. In summary, these experiments demonstrate the selective cytotoxicity and tumor suppressive ability of beta adrenergic inhibition on malignant vascular tumors and have laid the groundwork for a promising treatment of angiosarcomas in humans.

Growth Attenuation of Cutaneous Angiosarcoma With Propranolol-Mediated β-Blockade

IMPORTANCE Patients with stage T2 multilesion angiosarcomas of the scalp and face that are larger than 10 cm demonstrate a 2-year survival rate of 0%. To our knowledge, major therapeutic advances against this disease have not been reported for decades. Preclinical data indicate that blocking β-adrenergic signaling with propranolol hydrochloride disrupts angiosarcoma cell survival and xenograft angiosarcoma progression. OBSERVATIONS A patient presented with a β-adrenergic-positive multifocal stage T2 cutaneous angiosarcoma (≥20 cm) involving 80% of the scalp, left forehead, and left cheek, with no evidence of metastasis. The patient was immediately administered propranolol hydrochloride, 40 mg twice a day, as his workup progressed and treatment options were elucidated. Evaluation of the proliferative index of the tumor before and after only 1 week of propranolol monotherapy revealed a reduction in the proliferative index of the tumor by approximately 34%. A combination of propranolol hydrochloride, 40 mg 3 times a day, paclitaxel poliglumex, 2 mg/m2 infused weekly, and radiotherapy during the subsequent 8 months resulted in extensive tumor regression with no detectable metastases. CONCLUSIONS AND RELEVANCE Our data suggest that β-blockade alone substantially reduced angiosarcoma proliferation and, in combination with standard therapy, is effective for reducing the size of the tumor and preventing metastases. If successful, β-blockade could be the first major advancement in the treatment of angiosarcoma in decades.

Gene expression analysis reveals marked differences in the transcriptome of infantile hemangioma endothelial cells compared to normal dermal microvascular endothelial cells

BackgroundInfantile hemangiomas are benign vascular tumors primarily found on the skin in 10% of the pediatric population. The etiology of this disease is largely unknown and while large scale genomic studies have examined the transcriptomes of infantile hemangioma tumors as a whole, no study to date has compared the global gene expression profiles of pure infantile hemangioma endothelial cells (HEMECs) to that of normal human dermal microvascular endothelial cells (HDMVECs).MethodsTo shed light on the molecular differences between these normal and aberrant dermal endothelial cell types, we performed whole genome microarray analysis on purified cultures of HEMECs and HDMVECs. We then utilized qPCR and immunohistochemistry to confirm our microarray results.ResultsOur array analysis identified 125 genes whose expression was upregulated and 104 genes whose expression was downregulated by greater than two fold in HEMECs compared to HDMVECs. Bioinformatics analysis revealed three major classifications of gene functions that were altered in HEMECs including cell adhesion, cell cycle, and arachidonic acid production. Several of these genes have been reported to be critical regulators and/or mutated in cancer, vascular tumors, and vascular malformations. We confirmed the expression of a subset of these differentially expressed genes (ANGPT2, ANTXR1, SMARCE1, RGS5, CTAG2, LTBP2, CLDN11, and KISS1) using qPCR and utilized immunohistochemistry on a panel of paraffin embedded infantile hemangioma tumor tissues to demonstrate that the cancer/testis antigen CTAG2 is highly abundant in vessel-dense proliferating infantile hemangiomas and with significantly reduced levels during tumor involution as vascular density decreases.ConclusionOur data reveal that the transcriptome of HEMECs is reflective of a pro-proliferative cell type with altered adhesive characteristics. Moveover, HEMECs show altered expression of many genes that are important in the progression and prognosis of metastatic cancers.

Enrichment of the embryonic stem cell reprogramming factors Oct4, Nanog, Myc, and Sox2 in benign and malignant vascular tumors

BackgroundThe “stem cell theory of cancer” states that a subpopulation of cells with stem cell-like properties plays a central role in the formation, sustainment, spread, and drug resistant characteristics of malignant tumors. Recent studies have isolated distinct cell populations from infantile hemangiomas that display properties equivalent to aberrant progenitor cells, suggesting that, in addition to malignant tumors, benign tumors may also contain a stem cell-like component.MethodsIn this study, the expression levels of the embryonic stem cell reprogramming factors Oct4, Nanog, Myc, Sox2, and Klf4 were examined via immunohistochemistry in a panel of 71 benign, borderline, and malignant vascular tumors including capillary hemangioma, cavernous hemangioma, granulomatous hemangioma, venous hemangioma, hemangioendothelioma, hemangiopericytoma, and angiosarcoma. Antigenicity for each protein was quantified based on staining intensity and percentage of tissue positive for each antigen, and subsequently compared to data obtained from two control tissue sets: 10 vascular tissues and a panel of 58 various malignant sarcomas.Results and discussionWith the exception of Myc (which was only present in a subset of benign, borderline, and malignant tumors), Oct4, Nanog, Sox2, and Klf4 were detectable at variable levels across both normal and diseased tissues. Semi-quantitative evaluation of our immunohistochemical staining revealed that protein expression of Oct4, Nanog, Myc, and Sox2, but not Klf4, was significantly increased in benign, borderline, and malignant vascular tumors relative to non-diseased vascular tissue controls. Interestingly, the enhanced levels of Oct4, Nanog, Myc, and Sox2 protein were approximately equivalent between benign, borderline, and malignant vascular tumors.ConclusionsThese findings provide supporting evidence that enrichment for proteins involved in pluripotency is not restricted solely to malignant tumors as is suggested by the “stem cell theory of cancer”, but additionally extends to common benign vascular tumors such as hemangiomas.

A genomics approach to identify susceptibilities of breast cancer cells to " fever-range" hyperthermia

BackgroundPreclinical and clinical studies have shown for decades that tumor cells demonstrate significantly enhanced sensitivity to “fever range” hyperthermia (increasing the intratumoral temperature to 42-45°C) than normal cells, although it is unknown why cancer cells exhibit this distinctive susceptibility.MethodsTo address this issue, mammary epithelial cells and three malignant breast cancer lines were subjected to hyperthermic shock and microarray, bioinformatics, and network analysis of the global transcription changes was subsequently performed.ResultsBioinformatics analysis differentiated the gene expression patterns that distinguish the heat shock response of normal cells from malignant breast cancer cells, revealing that the gene expression profiles of mammary epithelial cells are completely distinct from malignant breast cancer lines following this treatment. Using gene network analysis, we identified altered expression of transcripts involved in mitotic regulators, histones, and non-protein coding RNAs as the significant processes that differed between the hyperthermic response of mammary epithelial cells and breast cancer cells. We confirmed our data via qPCR and flow cytometric analysis to demonstrate that hyperthermia specifically disrupts the expression of key mitotic regulators and G2/M phase progression in the breast cancer cells.ConclusionThese data have identified molecular mechanisms by which breast cancer lines may exhibit enhanced susceptibility to hyperthermic shock.

Morphological restriction of human coronary artery endothelial cells substantially impacts global gene expression patterns

Alterations in cell shape have been shown to modulate chromatin condensation and cell lineage specification; however, the mechanisms controlling these processes are largely unknown. Because endothelial cells experience cyclic mechanical changes from blood flow during normal physiological processes and disrupted mechanical changes as a result of abnormal blood flow, cell shape deformation and loss of polarization during coronary artery disease, we aimed to determine how morphological restriction affects global gene expression patterns. Human coronary artery endothelial cells (HCAECs) were cultured on spatially defined adhesive micropatterns, forcing them to conform to unique cellular morphologies differing in cellular polarization and angularity. We utilized pattern recognition algorithms and statistical analysis to validate the cytoskeletal pattern reproducibility and uniqueness of each micropattern, and performed microarray analysis on normal‐shaped and micropatterned HCAECs to determine how constrained cellular morphology affects gene expression patterns. Analysis of the data revealed that forcing HCAECs to conform to geometrically‐defined shapes significantly affects their global transcription patterns compared to nonrestricted shapes. Interestingly, gene expression patterns were altered in response to morphological restriction in general, although they were consistent regardless of the particular shape the cells conformed to. These data suggest that the ability of HCAECs to spread, although not necessarily their particular morphology, dictates their genomics patterns.

PD-1 and PD-L1 expression in bone and soft tissue sarcomas

PD-1 and its ligands have been shown to play a significant role in evasion of malignant tumour cells from the immune system. Last year, the Unites States Food and Drug Administration (FDA) approved anti-PD-1 inhibitors for treatment of non-small cell lung carcinoma and recently expanded the use of immunotherapy for metastatic urothelial cell carcinoma and Hodgkin lymphoma. However, studies on expression of PD-1 and its ligand in malignant bone and soft tissue sarcoma are sparse. In this study, we evaluated PD-1 and PD-L1 expression on variants of liposarcomas and rhabdomyosarcomas, osteosarcomas and chondrosarcomas. Tissue microarrays (TMAs) for liposarcomas (well differentiated, myxoid/round cell, and pleomorphic), rhabdomyosarcomas (alveolar, embryonal, pleomorphic, and spindle cell), conventional osteosarcomas and chondrosarcomas were stained for PD-1 and PD-L1 antibodies. Adipose tissue, skeletal muscle, bone, osteochondroma and lipoma were used as control and benign counterparts. Western blot was performed to evaluate expression of PD-1 and PD-L1 in four sarcoma cell lines. Osteosarcomas, chondrosarcomas, and all variants of liposarcomas and rhabdomyosarcomas over-expressed PD-1 relative to normal tissue. Expression of PD-1 in rhabdomyosarcomas was associated with higher tumour stage. Only one case of pleomorphic liposarcoma, one case of pleomorphic rhabdomyosarcoma and two cases of alveolar rhabdomyosarcomas were positive for PD-L1. Normal adipose tissue, skeletal muscle, and bone were negative for both PD-1 and PD-L1 and lipomas and osteochondroma weakly expressed PD-1 but not PD-L1. Western blot confirmed the presence of PD-1 protein in all four sarcoma cell lines. Overall, our results showed cytoplasmic expression of PD-1 in the bone and soft tissue sarcomas, while PD-L1 was negative. Whether these data are an indication for effectiveness of immunotherapy in the management of malignant bone and soft tissue sarcomas remains to be elucidated.

Enhanced expression of Programmed cell death 1 (PD-1) protein in benign vascular anomalies

Programmed cell death 1 (PD-1) and its ligands have been shown to play a significant role in evasion of malignant tumour cells from the immune system. Last year, the United States Food and Drug Administration (FDA) approved anti-PD-1 inhibitors for treatment of non-small cell lung carcinoma and recently has approved anti-PD-L1 blocker for treatment of metastatic urothelial cell carcinoma. However, the role that the immune system might have on benign tumours including vascular anomalies has received less attention. In this study, we evaluated PD-1 and PD-L1 expression on two benign vascular anomalies: infantile haemangiomas and venous malformations. Tissue microarrays (TMAs) from these two entities were stained for PD-1 and PD-L1 antibodies. Blood vessels from normal tissue were used as control. The endothelial cells in both infantile haemangioma and venous malformation showed high expression of PD-1 but were negative for PD-L1. Endothelial cells within the blood vessels in normal tissues were negative for both PD-1 and PD-L1. Our results showed over-expression of PD-1 in subsets of vascular anomalies, while PD-L1 was negative. This would raise the possibility of immunotherapy in benign vascular tumour when other options are exhausted.

Use of non-selective β-blockers is associated with decreased tumor proliferative indices in early stage breast cancer

Previous studies suggest beta-adrenergic receptor (β-AR) antagonists (β-blockers) decrease breast cancer progression, tumor metastasis, and patient mortality; however the mechanism for this is unknown. Immunohistochemical analysis of normal and malignant breast tissue revealed overexpression of β1-AR and β3-AR in breast cancer. A retrospective cross-sectional study of 404 breast cancer patients was performed to determine the effect of β-blocker usage on tumor proliferation. Our analysis revealed that non-selective β-blockers, but not selective β-blockers, reduced tumor proliferation by 66% (p < 0.0001) in early stage breast cancer compared to non-users. We tested the efficacy of propranolol on an early stage breast cancer patient, and quantified the tumor proliferative index before and after treatment, revealing a propranolol-mediated 23% reduction (p = 0.02) in Ki67 positive tumor cells over a three-week period. The anti-proliferative effects of β-blockers were measured in a panel of breast cancer lines, demonstrating that mammary epithelial cells were resistant to propranolol, and that most breast cancer cell lines displayed dose dependent viability decreases following treatment. Selective β-blockers alone or in combination were not as effective as propranolol at reducing breast cancer cell proliferation. Molecular analysis revealed that propranolol treatment of the SK-BR-3 breast cancer line, which showed high sensitivity to beta blockade, led to a reduction in Ki67 protein expression, decreased phosphorylation of the mitogenic signaling regulators p44/42 MAPK, p38 MAPK, JNK, and CREB, increased phosphorylation of the cell survival/apoptosis regulators AKT, p53, and GSK3β. In conclusion, use of non-selective β-blockers in patients with early stage breast cancer may lead to decreased tumor proliferation.