Publications by authors named "Adriana Haimovitz-Friedman"

56 Publications

Chemotherapy-induced acute vascular injury involves intracellular generation of ROS via activation of the acid sphingomyelinase pathway.

Cell Signal 2021 Jun 26;82:109969. Epub 2021 Feb 26.

Department of Radiation Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA. Electronic address:

Several categories of chemotherapy confer substantial risk for late-term vascular morbidity and mortality. In the present study, we aimed to investigate the mechanism of acute chemotherapy-induced vascular injury in normal tissues. Specifically, we looked at activation of the acid sphingomyelinase (ASMase)/ceramide pathway, which leads to generation of reactive oxygen species (ROS) and induction of oxidative stress that may result in vascular injury. In particular, we focused on two distinct drugs, doxorubicin (DOX) and cisplatin (CIS) and their effects on normal endothelial cells. In vitro, DOX resulted in increased ASMase activity, intra-cellular ROS production and induction of apoptosis. CIS treatment generated significantly reduced effects in endothelial cells. In-vivo, murine femoral arterial blood flow was measured in real-time, during and after DOX or CIS administration, using fluorescence optical imaging system. While DOX caused constriction of small vessels and disintegration of large vessels' wall, CIS induced minor vascular changes in arterial blood flow, correlating with the in vitro findings. These results demonstrate that DOX induces acute vascular injury by increased ROS production, via activation of ASMase/ceramide pathway, while CIS increases ROS production and its immediate extracellular translocation, without causing detectable acute vascular injury. Our findings may potentially lead to the development of new strategies to prevent long-term cardiovascular morbidity in cancer survivors.
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http://dx.doi.org/10.1016/j.cellsig.2021.109969DOI Listing
June 2021

Chemotherapeutic Agents-Induced Ceramide-Rich Platforms (CRPs) in Endothelial Cells and Their Modulation.

Methods Mol Biol 2021 ;2187:215-221

Department of Radiation Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.

The prevailing mechanism of action of chemotherapeutic drugs has been challenged by the role of ceramide, a second messenger, shown to induce apoptosis, differentiation, growth arrest, senescence, and autophagy in different cells (Chabner BA, Roberts TG Jr, Nat Rev Cancer 5:65-72, 2005; Jacobi J et al, Cell Signal 29:52-61, 2017; Rotolo J et al, J Clin Invest 122:1786-1790, 2012; Truman JP et al, PLoS One 5:e12310, 2010). Certain chemotherapeutic drugs activate the acid sphingomyelinase (ASMase)/ceramide pathway, generating ceramide in the tumor endothelium and this microvascular dysfunction is crucial for the tumor response. Ceramide has fusigenic properties and as such, when generated within the plasma membrane, initiates the oligomerization of ceramide-and cholesterol-rich domains in the outer leaflet of the plasma membrane, leading to the formation of ceramide-rich microdomains/platforms (CRP) (Jacobi J et al, Cell Signal 29:52-61, 2017; Truman JP et al, PLoS One 5:e12310, 2010; van Hell AJ et al, Cell Signal 34:86-91, 2017; Hajj C, Haimovitz-Friedman A, Handb Exp Pharmacol 216:115-130, 2013) known as "signaling platform." This chapter will discuss the generation, detection, and quantitation of CRP and their possible modulation in endothelial cells, in vitro and in vivo in response to certain chemotherapeutic drugs.
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http://dx.doi.org/10.1007/978-1-0716-0814-2_12DOI Listing
March 2021

Manipulating Oxidative Stress Following Ionizing Radiation.

J Cell Signal 2020 ;1(1):8-13

Department of Medicine, Renal Service, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.

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http://dx.doi.org/10.33696/signaling.1.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7299208PMC
January 2020

Photobiomodulation effects on head and neck squamous cell carcinoma (HNSCC) in an orthotopic animal model.

Support Care Cancer 2020 Jun 8;28(6):2721-2727. Epub 2019 Nov 8.

Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Background: Photobiomodulation (PBM) has shown efficacy in preventing and treating cancer therapy-induced mucositis and dermatitis. However, there is contradictory information regarding the effect of PBM on (pre)malignant cells, which has led to questions regarding the safety of this technique. We address this issue using an orthotopic mouse model (Cal-33) with human squamous cell carcinoma of the oral cavity.

Methods: Mice with actively growing orthotopic Cal-33 head and neck carcinoma tumors were divided into 4 groups: control, PBM only, radiation therapy (RT) only, and PBM + RT. We performed three experiments: (1) PBM at 660 nm, 18.4 J/cm, and 5 RT × 4 Gy doses delivered daily; (2) PBM at 660 nm, 18.4 J/cm, and 1 × 15 Gy RT; and (3) PBM at 660 nm + 850 nm, 45 mW/cm, 3.4 J/cm, and 1 × 15 Gy RT. Mice were weighed daily and tumor volumes were evaluated by IVIS. Survival time was also evaluated.

Results: Animals treated with RT survived significantly longer and had significantly smaller tumor volume when compared with the control and PBM-only treatment groups. No significant differences were noted between the RT alone and PBM + RT groups in any of the experiments.

Conclusion: Our results suggest that PBM at the utilized parameters does not provide protection to the tumor from the killing effects of RT.
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http://dx.doi.org/10.1007/s00520-019-05060-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7682488PMC
June 2020

Organoids Reveal That Inherent Radiosensitivity of Small and Large Intestinal Stem Cells Determines Organ Sensitivity.

Cancer Res 2020 03 5;80(5):1219-1227. Epub 2019 Nov 5.

Laboratory of Signal Transduction, Memorial Sloan Kettering Cancer Center, New York, New York.

Tissue survival responses to ionizing radiation are nonlinear with dose, rather yielding tissue-specific descending curves that impede straightforward analysis of biologic effects. Apoptotic cell death often occurs at low doses, while at clinically relevant intermediate doses, double-strand break misrepair yields mitotic death that determines outcome. As researchers frequently use a single low dose for experimentation, such strategies may inaccurately depict inherent tissue responses. Cutting edge radiobiology has adopted full dose survival profiling and devised mathematical algorithms to fit curves to observed data to generate highly reproducible numerical data that accurately define clinically relevant inherent radiosensitivities. Here, we established a protocol for irradiating organoids that delivers radiation profiles simulating the organ of origin. This technique yielded highly similar dose-survival curves of small and large intestinal crypts and their cognate organoids analyzed by the single-hit multi-target (SHMT) algorithm, outcomes reflecting the inherent radiation profile of their respective Lgr5 stem cell populations. As this technological advance is quantitative, it will be useful for accurate evaluation of intestinal (patho)physiology and drug screening. SIGNIFICANCE: These findings establish standards for irradiating organoids that deliver radiation profiles that phenocopy the organ of origin..
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http://dx.doi.org/10.1158/0008-5472.CAN-19-0312DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7056505PMC
March 2020

Immunomodulatory Effects of Stereotactic Body Radiation Therapy: Preclinical Insights and Clinical Opportunities.

Int J Radiat Oncol Biol Phys 2019 Mar 2. Epub 2019 Mar 2.

Department of Radiation Oncology and Molecular Radiation Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland.

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http://dx.doi.org/10.1016/j.ijrobp.2019.02.046DOI Listing
March 2019

Single-dose radiotherapy disables tumor cell homologous recombination via ischemia/reperfusion injury.

J Clin Invest 2019 02 14;129(2):786-801. Epub 2019 Jan 14.

Department of Radiation Oncology.

Tumor cure with conventional fractionated radiotherapy is 65%, dependent on tumor cell-autonomous gradual buildup of DNA double-strand break (DSB) misrepair. Here we report that single-dose radiotherapy (SDRT), a disruptive technique that ablates more than 90% of human cancers, operates a distinct dual-target mechanism, linking acid sphingomyelinase-mediated (ASMase-mediated) microvascular perfusion defects to DNA unrepair in tumor cells to confer tumor cell lethality. ASMase-mediated microcirculatory vasoconstriction after SDRT conferred an ischemic stress response within parenchymal tumor cells, with ROS triggering the evolutionarily conserved SUMO stress response, specifically depleting chromatin-associated free SUMO3. Whereas SUMO3, but not SUMO2, was indispensable for homology-directed repair (HDR) of DSBs, HDR loss of function after SDRT yielded DSB unrepair, chromosomal aberrations, and tumor clonogen demise. Vasoconstriction blockade with the endothelin-1 inhibitor BQ-123, or ROS scavenging after SDRT using peroxiredoxin-6 overexpression or the SOD mimetic tempol, prevented chromatin SUMO3 depletion, HDR loss of function, and SDRT tumor ablation. We also provide evidence of mouse-to-human translation of this biology in a randomized clinical trial, showing that 24 Gy SDRT, but not 3×9 Gy fractionation, coupled early tumor ischemia/reperfusion to human cancer ablation. The SDRT biology provides opportunities for mechanism-based selective tumor radiosensitization via accessing of SDRT/ASMase signaling, as current studies indicate that this pathway is tractable to pharmacologic intervention.
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http://dx.doi.org/10.1172/JCI97631DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6355243PMC
February 2019

An Antitumor Immune Response Is Evoked by Partial-Volume Single-Dose Radiation in 2 Murine Models.

Int J Radiat Oncol Biol Phys 2019 03 18;103(3):697-708. Epub 2018 Oct 18.

Department of Radiation Oncology, Memorial Sloan Kettering Cancer Center, New York, New York. Electronic address:

Purpose: This study examined tumor growth delay resulting from partial irradiation in preclinical mouse models.

Methods And Materials: We investigated 67NR murine orthotopic breast tumors in both immunocompetent and nude mice. Treatment was delivered to 50% or 100% of the tumor using a 2 × 2 cm collimator on a microirradiator. Radiation response was modulated by treatment with anti-CD8 and anti-intercellular adhesion molecule (anti-ICAM) antibodies. Similar experiments were performed using the less immunogenic Lewis lung carcinoma mouse model. Tumor growth delay and γ-H2AX phosphorylation were measured, and immune response was assessed by immunofluorescence and flow cytometry at 1 and 7 days after radiation therapy. Tumor expression of cellular adhesion molecules was also measured at different times after radiation therapy.

Results: Partial irradiation led to tumor responses similar to those of fully exposed tumors in immunocompetent mice, but not in nude mice. After a single dose of 10 Gy, infiltration of CD8 T cells was observed along with increased expression of ICAM. The response to 10 Gy in hemi-irradiated tumors was abrogated by treatment with either anti-CD8 or anti-ICAM antibodies. Similar responses were obtained in the less immunogenic Lewis lung carcinoma mouse model delivering 15 Gy to half the tumor volume. Treatment with FTY720, a compound that inhibits T-cell egress from lymph nodes, did not affect tumor response at the time of CD8 T cells infiltration in the nonirradiated area of the tumor. This result indicated that the most likely source of these cells is the irradiated portion of the hemi-irradiated tumors. In addition, a significant abscopal effect was observed after partial irradiation with a single dose of 10 Gy in the 67NR model.

Conclusions: In these models, radiation controls tumor growth both directly through cell killing and indirectly through immune activation. This outcome raises the possibility that this effect could be induced in the clinic.
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http://dx.doi.org/10.1016/j.ijrobp.2018.10.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6764416PMC
March 2019

An optical nanoreporter of endolysosomal lipid accumulation reveals enduring effects of diet on hepatic macrophages in vivo.

Sci Transl Med 2018 10;10(461)

Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.

The abnormal accumulation of lipids within the endolysosomal lumen occurs in many conditions, including lysosomal storage disorders, atherosclerosis, nonalcoholic fatty liver disease (NAFLD), and drug-induced phospholipidosis. Current methods cannot monitor endolysosomal lipid content in vivo, hindering preclinical drug development and research into the mechanisms linking endolysosomal lipid accumulation to disease progression. We developed a single-walled carbon nanotube-based optical reporter that noninvasively measures endolysosomal lipid accumulation via bandgap modulation of its intrinsic near-infrared emission. The reporter detected lipid accumulation in Niemann-Pick disease, atherosclerosis, and NAFLD models in vivo. By applying the reporter to the study of NAFLD, we found that elevated lipid quantities in hepatic macrophages caused by a high-fat diet persist long after reverting to a normal diet. The reporter dynamically monitored endolysosomal lipid accumulation in vivo over time scales ranging from minutes to weeks, indicating its potential to accelerate preclinical research and drug development processes.
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http://dx.doi.org/10.1126/scitranslmed.aar2680DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6543545PMC
October 2018

Phosphorylation state of Ser in α-tubulin is a toggle switch that controls proliferating human breast tumors.

Cell Signal 2018 12 1;52:74-82. Epub 2018 Sep 1.

Department of Chemistry & Biochemistry, Queens College, USA; Graduate Center of The City University of New York, USA. Electronic address:

Engineered overexpression of protein kinase Cα (PKCα) is known to phosphorylate Ser in α-tubulin resulting in stimulated microtubule dynamics and cell motility, and activation of an epithelial-mesenchymal transition (EMT) in non-transformed human breast cells. Here it is shown that endogenous phosphorylation of native α-tubulin in two metastatic breast cell lines, MDA-MB-231-LM2-4175 and MDA-MB-468 is detected at PKC phosphorylation sites. α-Tubulin mutants that simulated phosphorylated (S165D) or non-phosphorylated (S165 N) states were stably expressed in MDA-MB-231-LM2-4175 cells. The S165D-α-tubulin mutant engendered expression of the EMT biomarker N-cadherin, whereas S165 N-α-tubulin suppressed N-cadherin and induced E-cadherin expression, revealing a 'cadherin switch'. S165 N-α-tubulin engendered more rapid passage through the cell cycle, induced shorter spindle fibers and exhibited more rapid proliferation. In nude mice injected with MDA-MB-231-LM2-4175 cells, cells expressing S165 N-α-tubulin (but not the S165D mutant) produced hyper-proliferative lung tumors with increased tumor incidence and higher Ki67 expression. These results implicate the phosphorylation state of Ser in α-tubulin as a PKC-regulated molecular switch that causes breast cells to exhibit either EMT characteristics or hyper-proliferation. Evaluation of genomic databases of human tumors strengthens the clinical significance of these findings.
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http://dx.doi.org/10.1016/j.cellsig.2018.08.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6765385PMC
December 2018

Pazopanib radio-sensitization of human sarcoma tumors.

Oncotarget 2018 Feb 20;9(10):9311-9324. Epub 2018 Jan 20.

Department of Radiation Oncology, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Recent data in our laboratory indicate that engagement of host-derived microenvironmental elements impact tumor response to single high dose radiation therapy (SDRT). In these studies we showed that microvascular endothelial damage plays a critical role in tumor response as regulator of direct lethal damage of SDRT. Using a genetic model of Acid Sphingomyelinase (ASMase)-deficient mice we showed that activation of this enzyme by SDRT-induced damage in the endothelium is mandatory for tumor cure. ASMase activation triggers ceramide-mediated apoptosis, and therein microvascular dysfunction, which increased the vulnerability of tumor cells to lethal damage by radiation. Angiogenic factors repressed this activity while a monoclonal antibody targeting VEGF, de-repressed ASMase activity and radiosensitized tumor endothelium when delivered immediately prior to SDRT. In this study, we tested the effect of SDRT in combination with the short-acting anti-angiogenic agent, Pazopanib (anti-VEGFR-1/2/3, PDGF-α/β and c-kit), in two xenograft models of human sarcoma. Pre-treatment with a single dose of Pazopanib increased SDRT-induced ASMase activity and endothelial dysfunction and , enhancing SDRT tumor cure, and exhibiting critical dependence on timing relative to SDRT exposure, suggesting a mechanism of action identical to that demonstrated for anti-VEGF/VEGFR2 antibodies. These results demonstrate the ability of Pazopanib to shift the response towards tumor cure and could therefore have a significant impact on clinical trial development in combination with SDRT for sarcoma cancer patients.
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http://dx.doi.org/10.18632/oncotarget.24281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5823639PMC
February 2018

Logarithmic expansion of LGR5 cells in human colorectal cancer.

Cell Signal 2018 Jan 25;42:97-105. Epub 2017 Sep 25.

Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA. Electronic address:

Stem cells of the small and large intestine are marked by expression of the Wnt target gene LGR5, a leucine-rich-repeat-containing G protein-coupled receptor. Previous studies reported increased expression of LGR5 in human colorectal cancer (CRC) compared to normal tissue either by immunohistochemistry or in situ hybridization (ISH). However, as these studies were semi-quantitative they did not provide a numerical estimate of the magnitude of this effect. While we confirm that LGR5 cells are exclusively located at the base of normal human small and large intestinal crypts, representing approximately 6% of total crypt cells, we show this cell population is 10-fold expanded in all grades of CRC, representing as much as 70% of the cells of tumor crypt-like structures. This expansion of the LGR5 compartment coincides with maintenance of crypt-like glandular structure (adenomas, and well and moderately differentiated adenocarcinomas), and is reduced in poorly differentiated CRC, where crypt-like glandular architecture is lost, accompanied by reduced epithelial terminal differentiation. Altogether these results indicate that LGR5 cell expansion is a hallmark of CRC tumorigenesis occurring during progression to adenoma, supporting CRC as a stem cell disease with implications for CRC therapy.
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http://dx.doi.org/10.1016/j.cellsig.2017.09.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5766032PMC
January 2018

A Combination of Radiation and the Hypoxia-Activated Prodrug Evofosfamide (TH-302) is Efficacious against a Human Orthotopic Pancreatic Tumor Model.

Transl Oncol 2017 Oct 31;10(5):760-765. Epub 2017 Jul 31.

Department of Medical Physics, MSKCC, 1275 York Avenue, New York, NY, 10065.

This study was designed to investigate the effect of single-dose radiation therapy (RT) in combination with evofosfamide (TH-302), a hypoxia-activated prodrug, in a pre-clinical model of pancreatic cancer. AsPC1 tumors were implanted orthotopically in the pancreas of nude mice. Tumors were treated with 15 Gy of RT, using a 1 cm diameter field, and delivered as a continuous arc. Image-guidance to center the field on the tumor was based on CT imaging with intraperitoneal contrast. Evofosfamide (100 mg/kg, i.p.) was administered 3 hours before RT. Tumor volumes were measured using ultrasound, and regrowth curves were plotted. Tumor hypoxia and cell proliferation were measured using pimonidazole and the thymidine analog EdU, respectively. In vitro clonogenic assays were performed. Tumors were shown to contain substantial areas of hypoxia, as calculated by percent pimonidazole staining. Evofosfamide was active in these tumors, as demonstrated by a significant reduction in uptake of the thymidine analog EdU. This effect was visible in oxygenated tissue, consistent with the previously reported bystander effects of evofosfamide. RT produced significant regrowth delay, as did evofosfamide. The combination of both agents produced a growth delay that was at least equal to the sum of the two treatments given separately. The improvement in tumor response when evofosfamide is combined with RT supports the hypothesis that hypoxia is a cause of radioresistance in high dose RT for pancreatic cancer. Assessing the efficacy and safety of stereotactic radiation treatment and evofosfamide is warranted in patients with locally advanced pancreatic cancer.
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http://dx.doi.org/10.1016/j.tranon.2017.06.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5538966PMC
October 2017

In Vitro and In Vivo Comparison of Gemcitabine and the Gemcitabine Analog 1-(2'-deoxy-2'-fluoroarabinofuranosyl) Cytosine (FAC) in Human Orthotopic and Genetically Modified Mouse Pancreatic Cancer Models.

Mol Imaging Biol 2017 12;19(6):885-892

Department of Medical Physics, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Purpose: Although gemcitabine is a mainstay of pancreatic cancer therapy, it is only moderately effective, and it would be desirable to measure drug uptake in patients. 1-(2'-deoxy-2'-fluoroarabinofuranosyl) cytosine (FAC), is an analog of gemcitabine, and when labeled with F-18, it may be a potential surrogate PET tracer for the drug.

Procedures: [F]FAC was synthesized to a radiochemical purity of >96 %. The human tumor lines AsPC1, BxPC3, Capan-1, Panc1, and MiaPaca2 were grown orthotopically in nude mice. KPC mice that conditionally express oncogenic K-ras and p53 mutations in pancreatic tissue were also used. The intra-tumoral distributions of [C]gemcitabine and [F]FAC were mapped with autoradiography. The inter-tumor correlation between [C]gemcitabine and [F]FAC was established in the orthotopic tumors. Expression of the equilibrative and concentrative nucleoside transporters (ENT, CNT) in vitro was detected by western blotting. Drug uptake was characterized in vitro using [H]gemcitabine and the effect of transporter inhibition on gemcitabine and FAC uptake was investigated. The relative affinity of cells for gemcitabine and FAC was tested in competition assays. The cell lines differed in sensitivity to transport inhibitors and in competition studies. There was a good in vivo correlation between the total uptake of [F]FAC and [C]gemcitabine, measured across all orthotopic tumors. Using the KPC and BxPC3 models, we found that [C]gemcitabine and [F]FAC were largely co-localized.

Conclusions: In the lines examined here, [F]FAC uptake correlates well with gemcitabine in vivo, supporting the notion that [F]FAC can serve as a PET radiotracer surrogate to determine the uptake and distribution of gemcitabine within pancreatic tumors.
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http://dx.doi.org/10.1007/s11307-017-1078-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5696795PMC
December 2017

Gemcitabine kills proliferating endothelial cells exclusively via acid sphingomyelinase activation.

Cell Signal 2017 06 24;34:86-91. Epub 2017 Feb 24.

Laboratory of Signal Transduction, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA. Electronic address:

Gemcitabine is a widely-used anti-cancer drug with a well-defined mechanism of action in normal and transformed epithelial cells. However, its effect on endothelial cells is largely unknown. Acid sphingomyelinase (ASMase) is highly expressed in endothelial cells, converting plasma membrane sphingomyelin to pro-apoptotic ceramide upon activation by diverse stresses. In the current study, we investigated gemcitabine impact in primary cultures of endothelial cells. We find baseline ASMase increases markedly in bovine aortic endothelial cells (BAEC) as they transit from a proliferative to a confluent growth-arrested state. Further, gemcitabine activates ASMase and induces release of a secretory ASMase form into the media only in proliferating endothelial cells. Additionally, proliferative, but not growth-arrested BAEC, are sensitive to gemcitabine-induced apoptotic death, an effect blocked by inhibiting ASMase with imipramine or by binding ceramide on the cell surface with an anti-ceramide Ab. Confluent growth-arrested BAEC can be re-sensitized to gemcitabine-induced apoptosis by provision of exogenous sphingomyelinase. A highly similar phenotype was observed in primary cultures of human coronary artery endothelial cells. These findings reveal a previously-unrecognized mechanism of gemcitabine cytotoxicity in endothelium that may well contribute to its clinical benefit, and suggest the potential for further improvement of its clinical efficacy via pharmacologic modulation of ASMase/ceramide signaling in proliferative tumor endothelium.
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http://dx.doi.org/10.1016/j.cellsig.2017.02.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5565230PMC
June 2017

Distinct Levels of Radioresistance in Lgr5 Colonic Epithelial Stem Cells versus Lgr5 Small Intestinal Stem Cells.

Cancer Res 2017 04 15;77(8):2124-2133. Epub 2017 Feb 15.

Laboratory of Signal Transduction, Memorial Sloan Kettering Cancer Center, New York, New York.

Although small and large intestines possess seemingly similar Wnt-driven leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) adult epithelial stem cells, we report here that the two organs exhibit distinct mechanisms of tissue response to ionizing radiation. Employing transgenic mice and Lgr5 hybridization, we found colonic epithelial stem cells (CESC) markedly more radioresistant than small intestinal crypt base columnar stem cells (CBC; D = 6.0 ± 0.3 Gy vs. 1.3 ± 0.1, respectively; < 0.01). Accordingly, CESCs survived 30 Gy exposure, while CBCs were completely depleted after 15 Gy. EdU incorporation studies indicated that after 19 Gy, CBCs exited growth arrest at 12 hours, resuming normal mitotic activity despite 60% of this population displaying residual γH2AX foci, indicative of persistent unrepaired DNA damage. Checkpoint recovery before complete double-strand break (DSB) repair represents the of a newly defined potentially lethal pathophysiology termed checkpoint adaptation. In the small intestinal mucosa, checkpoint adaptation resulted in CBCs succumbing to an 8-fold increase in the incidence of highly lethal chromosomal aberrations and mitotic catastrophe by 48 hours postradiation. In contrast, Lgr5 CESCs displayed delayed checkpoint recovery at 48 hours post-19 Gy, coordinated with complete DSB repair and regeneration of colonic mucosa originating, at least in part, from surviving CESCs. The discovery that small intestinal CBCs succumb to checkpoint adaptation is the first demonstration that this aberrant cell-cycle response may drive mammalian tissue radiosensitivity. .
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http://dx.doi.org/10.1158/0008-5472.CAN-15-2870DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5621135PMC
April 2017

Tumour-specific PI3K inhibition via nanoparticle-targeted delivery in head and neck squamous cell carcinoma.

Nat Commun 2017 02 13;8:14292. Epub 2017 Feb 13.

Human Oncology &Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA.

Alterations in PIK3CA, the gene encoding the p110α subunit of phosphatidylinositol 3-kinase (PI3Kα), are frequent in head and neck squamous cell carcinomas. Inhibitors of PI3Kα show promising activity in various cancer types, but their use is curtailed by dose-limiting side effects such as hyperglycaemia. In the present study, we explore the efficacy, specificity and safety of the targeted delivery of BYL719, a PI3Kα inhibitor currently in clinical development in solid tumours. By encapsulating BYL719 into P-selectin-targeted nanoparticles, we achieve specific accumulation of BYL719 in the tumour milieu. This results in tumour growth inhibition and radiosensitization despite the use of a sevenfold lower dose of BYL719 compared with oral administration. Furthermore, the nanoparticles abrogate acute and chronic metabolic side effects normally observed after BYL719 treatment. These findings offer a novel strategy that could potentially enhance the efficacy of PI3Kα inhibitors while mitigating dose-limiting toxicity in patients with head and neck squamous cell carcinomas.
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http://dx.doi.org/10.1038/ncomms14292DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5316830PMC
February 2017

Targeting acid sphingomyelinase with anti-angiogenic chemotherapy.

Cell Signal 2017 01 1;29:52-61. Epub 2016 Oct 1.

Department of Radiation Oncology, USA. Electronic address:

Despite great promise, combining anti-angiogenic and conventional anti-cancer drugs has produced limited therapeutic benefit in clinical trials, presumably because mechanisms of anti-angiogenic tissue response remain only partially understood. Here we define a new paradigm, in which anti-angiogenic drugs can be used to chemosensitize tumors by targeting the endothelial acid sphingomyelinase (ASMase) signal transduction pathway. We demonstrate that paclitaxel and etoposide, but not cisplatin, confer ASMase-mediated endothelial injury within minutes. This rapid reaction is required for human HCT-116 colon cancer xenograft complete response and growth delay. Whereas VEGF inhibits ASMase, anti-VEGFR2 antibodies de-repress ASMase, enhancing endothelial apoptosis and drug-induced tumor response in asmase, but not in asmase, hosts. Such chemosensitization occurs only if the anti-angiogenic drug is delivered 1-2h before chemotherapy, but at no other time prior to or post chemotherapy. Our studies suggest that precisely-timed administration of anti-angiogenic drugs in combination with ASMase-targeting anti-cancer drugs is likely to optimize anti-tumor effects of systemic chemotherapy. This strategy warrants evaluation in future clinical trials.
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http://dx.doi.org/10.1016/j.cellsig.2016.09.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5138150PMC
January 2017

Radiation-Induced Microvascular Injury as a Mechanism of Salivary Gland Hypofunction and Potential Target for Radioprotectors.

Radiat Res 2016 08 26;186(2):189-95. Epub 2016 Jul 26.

c Radiation Oncology, Memorial Sloan-Kettering Cancer Center, New York, New York;

Radiation therapy is commonly used to treat patients with head and neck squamous cell carcinoma (HNSCC). One of the major side effects of radiotherapy is injury to the salivary glands (SG), which is thought to be mediated by microvascular dysfunction leading to permanent xerostomia. The goal of this study was to elucidate the mechanism of radiation-induced microvasculature damage and its impact on SG function. We measured bovine aortic endothelial cell (BAEC) apoptosis and ceramide production in response to 5 Gy irradiation, either alone or with reactive oxygen species (ROS) scavengers. We then investigated the effect of a single 15 Gy radiation dose on murine SG function. BAECs exposed to 5 Gy underwent apoptosis with increased ceramide production, both prevented by ROS scavengers. Among the 15 Gy irradiated mice, there was considerable weight loss, alopecia and SG hypofunction manifested by reduced saliva production and lower lysozyme levels. All of these effects, except for the lysozyme levels, were prevented by pretreatment with ROS scavengers. Microvessel density was significantly lower in the SG of irradiated mice compared to the control group, and this effect was significantly attenuated by pretreatment with Tempol. This study demonstrates that radiation-induced SG hypofunction is to a large extent mediated by microvascular dysfunction involving ceramide and ROS generation. These findings strongly suggest that ROS scavengers may serve as potential radioprotectors of SG function in patients undergoing radiotherapy for HNSCC.
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http://dx.doi.org/10.1667/RR14431.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5134326PMC
August 2016

P-selectin is a nanotherapeutic delivery target in the tumor microenvironment.

Sci Transl Med 2016 06;8(345):345ra87

Molecular Pharmacology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Weill Cornell Medical College, New York, NY 10065, USA.

Disseminated tumors are poorly accessible to nanoscale drug delivery systems because of the vascular barrier, which attenuates extravasation at the tumor site. We investigated P-selectin, a molecule expressed on activated vasculature that facilitates metastasis by arresting tumor cells at the endothelium, for its potential to target metastases by arresting nanomedicines at the tumor endothelium. We found that P-selectin is expressed on cancer cells in many human tumors. To develop a targeted drug delivery platform, we used a fucosylated polysaccharide with nanomolar affinity to P-selectin. The nanoparticles targeted the tumor microenvironment to localize chemotherapeutics and a targeted MEK (mitogen-activated protein kinase kinase) inhibitor at tumor sites in both primary and metastatic models, resulting in superior antitumor efficacy. In tumors devoid of P-selectin, we found that ionizing radiation guided the nanoparticles to the disease site by inducing P-selectin expression. Radiation concomitantly produced an abscopal-like phenomenon wherein P-selectin appeared in unirradiated tumor vasculature, suggesting a potential strategy to target disparate drug classes to almost any tumor.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5064151PMC
http://dx.doi.org/10.1126/scitranslmed.aaf7374DOI Listing
June 2016

Targeting Homologous Recombination in Notch-Driven C. elegans Stem Cell and Human Tumors.

PLoS One 2015 29;10(6):e0127862. Epub 2015 Jun 29.

Laboratory of Signal Transduction, Memorial Sloan Kettering Cancer Center, New York, New York, United States of America.

Mammalian NOTCH1-4 receptors are all associated with human malignancy, although exact roles remain enigmatic. Here we employ glp-1(ar202), a temperature-sensitive gain-of-function C. elegans NOTCH mutant, to delineate NOTCH-driven tumor responses to radiotherapy. At ≤20°C, glp-1(ar202) is wild-type, whereas at 25°C it forms a germline stem cell⁄progenitor cell tumor reminiscent of human cancer. We identify a NOTCH tumor phenotype in which all tumor cells traffic rapidly to G2⁄M post-irradiation, attempt to repair DNA strand breaks exclusively via homology-driven repair, and when this fails die by mitotic death. Homology-driven repair inactivation is dramatically radiosensitizing. We show that these concepts translate directly to human cancer models.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0127862PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4485896PMC
March 2016

Novel mechanisms of action of classical chemotherapeutic agents on sphingolipid pathways.

Biol Chem 2015 Jun;396(6-7):669-79

The prevailing mechanisms of action of traditional chemotherapeutic agents have been challenged by sphingolipid cancer research. Many studies have shown that ceramide generation in response to cytotoxic agents is central to tumor cell death. Ceramide can be generated either via hydrolysis of cell-membrane sphingomyelin by sphingomyelinases, hydrolysis of cerebrosides, or via de novo synthesis by ceramide synthases. Ceramide can act as a second messenger for apoptosis, senescence or autophagy. Inherent or acquired alterations in the sphingolipid pathway can account for resistance to the classic chemotherapeutic agents. In particular, it has been shown that activation of the acid ceramidase can lead to the formation of sphingosine 1-phosphate, which then antagonizes ceramide signaling by initiating a pro-survival signaling pathway. Furthermore, ceramide glycosylation catalyzed by glucosylceramide synthase converts ceramide to glucosylceramide, thus eliminating ceramide and consequently protecting cancer cells from apoptosis. In this review, we describe the effects of some of the most commonly used chemotherapeutic agents on ceramide generation, with a particular emphasis on strategies used to enhance the efficacy of these agents.
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http://dx.doi.org/10.1515/hsz-2014-0302DOI Listing
June 2015

Ceramide synthase inhibitor fumonisin B1 inhibits apoptotic cell death in SCC17B human head and neck squamous carcinoma cells after Pc4 photosensitization.

Photochem Photobiol Sci 2014 Nov;13(11):1621-7

Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI 48201, USA.

The sphingolipid ceramide modulates stress-induced cell death and apoptosis. We have shown that ceramide generated via de novo sphingolipid biosynthesis is required to initiate apoptosis after photodynamic therapy (PDT). The objective of this study was to define the role of ceramide synthase (CERS) in PDT-induced cell death and apoptosis using fumonisin B1 (FB), a CERS inhibitor. We used the silicon phthalocyanine Pc4 for PDT, and SCC17B cells, as a clinically-relevant model of human head and neck squamous carcinoma. zVAD-fmk, a pan-caspase inhibitor, as well as FB, protected cells from death after PDT. In contrast, ABT199, an inhibitor of the anti-apoptotic protein Bcl2, enhanced cell killing after PDT. PDT-induced accumulation of ceramide in the endoplasmic reticulum and mitochondria was inhibited by FB. PDT-induced Bax translocation to the mitochondria and cytochrome c release were also inhibited by FB. These novel data suggest that PDT-induced cell death via apoptosis is CERS/ceramide-dependent.
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http://dx.doi.org/10.1039/c4pp00292jDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4276310PMC
November 2014

Axitinib sensitization of high Single Dose Radiotherapy.

Radiother Oncol 2014 Apr 29;111(1):88-93. Epub 2014 Apr 29.

Laboratory of Signal Transduction, Memorial Sloan-Kettering Cancer Center, New York, USA.

Background And Purpose: Single Dose Radiation Therapy (SDRT) provides remarkably high rates of control even for tumors resistant to fractionated radiotherapy. SDRT tumor control depends on acute acid sphingomyelinase-mediated endothelial cell injury and monoclonal antibodies targeting Vascular Endothelial Cell Growth Factor (VEGF) signaling radiosensitized tumor endothelium when delivered immediately prior to irradiation. Here we evaluate the ability of the oral VEGF receptor inhibitor, axitinib, to sensitize tumor endothelium and increase tumor control with SDRT.

Methods And Materials: Axitinib was added to primary cultured endothelial cells, or administered orally to Sv129/BL6 mice bearing radiosensitive MCA/129 sarcoma or radioresistant B16F1 melanoma flank tumors, followed by SDRT. Endothelial apoptosis was assessed by TUNEL assay or bis-benzamide staining. Mice with irradiated tumors were followed for 90days to evaluate the impact of axitinib on SDRT tumor control.

Results: Pre-treatment with axitinib increased acute endothelial cell apoptosis following SDRT in vitro, and in vivo for both MCA/129 and B16F1 tumors. Axitinib correspondingly increased SDRT tumor growth delay and complete response rate (by 40%) for both tumors. Administration precisely 1h before SDRT was critical for radiosensitization.

Conclusions: Axitinib radiosensitizes tumor endothelial cells and enhances tumor cure with SDRT, which may permit dose de-escalation and significantly expand the range of clinical indications for SDRT.
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http://dx.doi.org/10.1016/j.radonc.2014.02.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4278650PMC
April 2014

Adenoviral transduction of human acid sphingomyelinase into neo-angiogenic endothelium radiosensitizes tumor cure.

PLoS One 2013 2;8(8):e69025. Epub 2013 Aug 2.

Laboratory of Signal Transduction, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America.

These studies define a new mechanism-based approach to radiosensitize tumor cure by single dose radiotherapy (SDRT). Published evidence indicates that SDRT induces acute microvascular endothelial apoptosis initiated via acid sphingomyelinase (ASMase) translocation to the external plasma membrane. Ensuing microvascular damage regulates radiation lethality of tumor stem cell clonogens to effect tumor cure. Based on this biology, we engineered an ASMase-producing vector consisting of a modified pre-proendothelin-1 promoter, PPE1(3x), and a hypoxia-inducible dual-binding HIF-2α-Ets-1 enhancer element upstream of the asmase gene, inserted into a replication-deficient adenovirus yielding the vector Ad5H2E-PPE1(3x)-ASMase. This vector confers ASMase over-expression in cycling angiogenic endothelium in vitro and within tumors in vivo, with no detectable enhancement in endothelium of normal tissues that exhibit a minute fraction of cycling cells or in non-endothelial tumor or normal tissue cells. Intravenous pretreatment with Ad5H2E-PPE1(3x)-ASMase markedly increases SDRT cure of inherently radiosensitive MCA/129 fibrosarcomas, and converts radiation-incurable B16 melanomas into biopsy-proven tumor cures. In contrast, Ad5H2E-PPE1(3x)-ASMase treatment did not impact radiation damage to small intestinal crypts as non-dividing small intestinal microvessels did not overexpress ASMase and were not radiosensitized. We posit that combination of genetic up-regulation of tumor microvascular ASMase and SDRT provides therapeutic options for currently radiation-incurable human tumors.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0069025PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3732255PMC
April 2014

Sphingolipids' role in radiotherapy for prostate cancer.

Handb Exp Pharmacol 2013 (216):115-30

Department of Radiation Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.

There are several well-established mechanisms involved in radiation-induced cell death in mammalian cell systems. The p53-mediated apoptotic pathway is the most widely recognized mechanism (Lowe et al. Nature 362:847-849, 1993), although apoptosis has long been considered a less relevant mechanism of radiation-induced cell death (Steel, Acta Oncol 40:968-975, 2001; Brown and Wouters, Cancer Res 59:1391-1399, 1999; Olive and Durand, Int J Radiat Biol 71:695-707, 1997). We and others have recently focused instead on the emerging links between radiation, apoptosis, and ceramide and showed that ceramide is a sphingolipid-derived second messenger capable of initiating apoptotic cascades in response to various stress stimuli, including radiation.Ceramide, the backbone of all sphingolipids, is synthesized by a family of ceramide synthases (CerS), each using acyl-CoAs of defined chain length for N-acylation of the sphingoid long-chain base. Six mammalian CerS homologs have been cloned that demonstrated high selectivity towards acyl-CoAs (Lahiri et al. FEBS Lett 581:5289-5294, 2007), and more recently, it was shown that their activity can be modulated by dimer formation (Mesicek et al. Cell Signal 22:1300-1307, 2010; Laviad et al. J Biol Chem 283:5677-5684, 2008).This de novo ceramide synthesis has been observed in irradiated cells through a pathway normally suppressed by ataxia telangiectasia-mutated (ATM) protein, a key component of the cellular response to DNA double-strand breaks (Liao et al. J Biol Chem 274:17908-17917, 1999). ATM is not the sole factor known to affect apoptotic potential by modulating CerS activity. Recent work has also implicated protein kinase Cα (PKCα) as a potential CerS activator (Truman et al. Cancer Biol Ther 8:54-63, 2009).In this review, we summarize involvement of CerS in sphingolipid-mediated apoptosis in irradiated human prostate cancer cells and discuss future directions in this field.
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http://dx.doi.org/10.1007/978-3-7091-1511-4_6DOI Listing
July 2013

Crypt base columnar stem cells in small intestines of mice are radioresistant.

Gastroenterology 2012 Nov 27;143(5):1266-1276. Epub 2012 Jul 27.

Laboratory of Signal Transduction, Memorial Sloan-Kettering Cancer Center, New York, New York. Electronic address:

Background & Aims: Adult stem cells have been proposed to be quiescent and radiation resistant, repairing DNA double-strand breaks by nonhomologous end joining. However, the population of putative small intestinal stem cells (ISCs) at position +4 from the crypt base contradicts this model, in that they are highly radiosensitive. Cycling crypt base columnar cells (CBCs) at crypt positions +1-3 recently were defined as an alternative population of ISCs. Little is known about the sensitivity of this stem cell population to radiation.

Methods: Radiation-induced lethality of CBCs was quantified kinetically in Lgr5-lacZ transgenic mice. γ-H2AX, BRCA1, RAD51, and DNA-PKcs foci were used as DNA repair surrogates to investigate the inherent ability of CBCs to recognize and repair double-strand breaks. 5-ethynyl-2'-deoxyuridine and 5-bromo-2'-deoxyuridine incorporation assays were used to study patterns of CBC growth arrest and re-initiation of cell cycling. Apoptosis was evaluated by caspase-3 staining.

Results: CBCs are relatively radioresistant, repairing DNA by homologous recombination significantly more efficiently than transit amplifying progenitors or villus cells. CBCs undergo apoptosis less than 24 hours after irradiation (32% ± 2% of total lethality) or mitotic death at 24-48 hours. Survival of CBCs at 2 days predicts crypt regeneration at 3.5 days and lethality from gastrointestinal syndrome. Crypt repopulation originates from CBCs that survive irradiation.

Conclusions: Adult ISCs in mice can cycle rapidly yet still be radioresistant. Importantly, homologous recombination can protect adult stem cell populations from genotoxic stress. These findings broaden and refine concepts of the phenotype of adult stem cells.
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http://dx.doi.org/10.1053/j.gastro.2012.07.106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3480544PMC
November 2012

Anti-ceramide antibody prevents the radiation gastrointestinal syndrome in mice.

J Clin Invest 2012 May 2;122(5):1786-90. Epub 2012 Apr 2.

Laboratory of Signal Transduction, Memorial Sloan-Kettering Cancer Center (MSKCC), New York, New York, USA.

Radiation gastrointestinal (GI) syndrome is a major lethal toxicity that may occur after a radiation/nuclear incident. Currently, there are no prophylactic countermeasures against radiation GI syndrome lethality for first responders, military personnel, or remediation workers entering a contaminated area. The pathophysiology of this syndrome requires depletion of stem cell clonogens (SCCs) within the crypts of Lieberkühn, which are a subset of cells necessary for postinjury regeneration of gut epithelium. Recent evidence indicates that SCC depletion is not exclusively a result of DNA damage but is critically coupled to ceramide-induced endothelial cell apoptosis within the mucosal microvascular network. Here we show that ceramide generated on the surface of endothelium coalesces to form ceramide-rich platforms that transmit an apoptotic signal. Moreover, we report the generation of 2A2, an anti-ceramide monoclonal antibody that binds to ceramide to prevent platform formation on the surface of irradiated endothelial cells of the murine GI tract. Consequently, we found that 2A2 protected against endothelial apoptosis in the small intestinal lamina propria and facilitated recovery of crypt SCCs, preventing the death of mice from radiation GI syndrome after high radiation doses. As such, we suggest that 2A2 represents a prototype of a new class of anti-ceramide therapeutics and an effective countermeasure against radiation GI syndrome mortality.
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http://dx.doi.org/10.1172/JCI59920DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3336980PMC
May 2012