David R. Soto-Pantoja

Angiotensin-(1-7) Inhibits Growth of Human Lung Adenocarcinoma Xenografts in Nude Mice through a Reduction in Cyclooxygenase-2

Angiotensin-(1-7) [Ang-(1-7)] is an endogenous peptide of the renin-angiotensin system with vasodilator and antiproliferative properties. Our previous studies showed that Ang-(1-7) reduced serum-stimulated growth of human lung cancer cells in vitro through activation of a unique AT((1-7)) receptor. The current study investigates the effect of Ang-(1-7) on lung tumor growth in vivo, using a human lung tumor xenograft model. Athymic mice with tumors resulting from injection of A549 human lung cancer cells were treated for 28 days with either i.v. saline or Ang-(1-7), delivered by implanted osmotic mini-pumps. Treatment with Ang-(1-7) reduced tumor volume by 30% compared with the size before treatment; in contrast, tumor size in the saline-treated animals increased 2.5-fold. These results correlate with a reduction in the proliferation marker Ki67 in the Ang-(1-7)-infused tumors when compared with the saline-infused tumor tissues. Treatment with Ang-(1-7) significantly reduced cyclooxygenase-2 (COX-2) mRNA and protein in tumors of Ang-(1-7)-infused mice when compared with mice treated with saline as well as in the parent A549 human lung cancer cells in tissue culture. These results suggest that Ang-(1-7) may decrease COX-2 activity and proinflammatory prostaglandins to inhibit lung tumor growth. In contrast, the heptapeptide had no effect on COX-1 mRNA in xenograft tumors or A549 cells. Because Ang-(1-7), a peptide with antithrombotic properties, reduces growth through activation of a selective AT((1-7)) receptor, our results suggest that the heptapeptide represents a novel treatment for lung cancer by reducing COX-2.

Radioprotection in Normal Tissue and Delayed Tumor Growth by Blockade of CD47 Signaling

Inhibition of a membrane receptor protects normal tissue from radiation injury and simultaneously enhances the ability of ionizing radiation to delay tumor growth. One major caveat of radiation therapy in cancer treatment is that the effective dose delivered to the individual is oftentimes necessarily limited to avoid major side effects that arise from collateral damage inflicted on surrounding normal tissue. Efforts to devise methods to sensitize tumor tissue to radiation injury or to protect normal tissue by scavenging for reactive by-products of radiation have only been moderately successful, as their broad clinical utility is hampered by a lack of specificity or by toxicity. Now, a team of researchers describes an approach to protecting normal human cells from high-dose radiation damage while, at the same time, increasing the sensitivity of tumor cells to radiation. We know that the pathway triggered by the secretory glycoprotein thrombospondin-1 (TSP1) and its corresponding membrane receptor CD47 in response to injury limits the survival of vascular cells and tissues. In this work, Roberts et al. have devised a strategy by which the systemic blockade of TSP1 and its receptor CD47 inhibits downstream signaling functions to protect against radiation injury the highly radiation-sensitive endothelial cells that line the lumen of the vasculature. More important, they show in mice injected with human tumors that suppression of CD47 by systemic administration of an antisense CD47 morpholino can sensitize the tumors to high-dose radiation therapy while keeping collateral damage at bay, exemplified in part by the resilience of the skin, muscle, and bone to radiation injury. Their experiments simultaneously show a significant delay in the time that these tumors take to grow back. This phenomenon remains to be explained, but there is an improvement in vascular function in irradiated, CD47-suppressed mice, and, at the cellular level, in vivo stem cells are still viable and proliferate, whereas peripheral immune cells, which infiltrate the inflammatory tumor microenvironment, are protected and recruited to the site. Although it remains speculative how CD47 participates in antitumor immunity within this experimental context, combined treatment of high-dose radiation and CD47 suppression in these translational studies suggests that a more aggressive therapeutic irradiation strategy with concurrent protection of neighboring normal tissue is possible. Testing the effectiveness of such a treatment strategy will be required to see whether this approach is useful. Radiation-induced damage of normal tissues restricts the therapeutic doses of ionizing radiation that can be delivered to tumors and thereby limits the effectiveness of radiotherapy. Thrombospondin-1 signaling through its cell surface receptor CD47 limits recovery from several types of stress, and mice lacking either gene are profoundly resistant to radiation injury. We describe strategies to protect normal tissues from radiation damage with antibodies to CD47 or thrombospondin-1, a CD47-binding peptide, or antisense suppression of CD47. A morpholino oligonucleotide targeting CD47 confers radioresistance to human endothelial cells in vitro and protects soft tissue, bone marrow, and tumor-associated leukocytes in irradiated mice. In contrast, CD47 suppression in mice bearing melanoma or squamous lung tumors before irradiation results in 89% and 71% smaller tumors, respectively. Thus, inhibition of CD47 signaling maintains the viability of normal tissues after irradiation while increasing the radiosensitivity of tumors.

Angiotensin-(1-7) inhibits tumor angiogenesis in human lung cancer xenografts with a reduction in vascular endothelial growth factor

Angiotensin-(1-7) [Ang-(1-7)] is an endogenous seven-amino acid peptide hormone with antiproliferative properties. Our previous studies showed that Ang-(1-7) inhibits the growth of human lung cancer cells in vitro and reduces the size of human lung tumor xenografts in vivo. In the current study, s.c. injection of Ang-(1-7) not only caused a significant reduction in human A549 lung tumor growth but also markedly decreased vessel density, suggesting that the heptapeptide inhibits angiogenesis to reduce tumor size. A decrease in human endothelial cell tubule formation in Matrigel was observed following a 16 h incubation with Ang-(1-7), with a maximal reduction at a 10 nmol/L concentration. Ang-(1-7) had similar antiangiogenic effects in the chick chorioallantoic membrane, causing a >50% decrease in neovascularization. The Ang-(1-7)-induced reduction in both endothelial cell tubule formation and vessel formation in the chick was completely blocked by the specific Ang-(1-7) receptor antagonist [d-proline7]-Ang-(1-7), suggesting that these biological actions are mediated by an AT(1-7) receptor. Ang-(1-7) significantly reduced vascular endothelial growth factor-A protein and mRNA in tumors from mice treated with the heptapeptide compared with saline controls as well as in the parent A549 human lung cancer cells in culture. These results suggest that Ang-(1-7) may attenuate tumor angiogenesis by reducing vascular endothelial growth factor-A, a primary proangiogenic protein. Taken together, this study shows that Ang-(1-7) exhibits significant antiangiogenic activity and may be a novel therapeutic agent for lung cancer treatment targeting a specific AT(1-7) receptor. [Mol Cancer Ther 2009;8(6):1676–83]

Thrombospondin-1 Signaling through CD47 Inhibits Self-renewal by Regulating c-Myc and Other Stem Cell Transcription Factors

Signaling through the thrombospondin-1 receptor CD47 broadly limits cell and tissue survival of stress, but the molecular mechanisms are incompletely understood. We now show that loss of CD47 permits sustained proliferation of primary murine endothelial cells, increases asymmetric division, and enables these cells to spontaneously reprogram to form multipotent embryoid body-like clusters. c-Myc, Klf4, Oct4, and Sox2 expression is elevated in CD47-null endothelial cells, in several tissues of CD47- and thrombospondin-1-null mice, and in a human T cell line lacking CD47. CD47 knockdown acutely increases mRNA levels of c-Myc and other stem cell transcription factors in cells and in vivo, whereas CD47 ligation by thrombospondin-1 suppresses c-Myc expression. The inhibitory effects of increasing CD47 levels can be overcome by maintaining c-Myc expression and are absent in cells with dysregulated c-Myc. Thus, CD47 antagonists enable cell self-renewal and reprogramming by overcoming negative regulation of c-Myc and other stem cell transcription factors.

CD47 deficiency confers cell and tissue radioprotection by activation of autophagy.

Accidental or therapeutic exposure to ionizing radiation has severe physiological consequences and can result in cell death. We previously demonstrated that deficiency or blockade of the ubiquitously expressed receptor CD47 results in remarkable cell and tissue protection against ischemic and radiation stress. Antagonists of CD47 or its ligand THBS1/thrombospondin 1 enhance cell survival and preserve their proliferative capacity. However the signaling pathways that mediate this cell-autonomous radioprotection are unclear. We now report a marked increase in autophagy in irradiated T-cells and endothelial cells lacking CD47. Irradiated T cells lacking CD47 exhibit significant increases in formation of autophagosomes comprising double-membrane vesicles visualized by electron microscopy and numbers of MAP1LC3A/B+ puncta. Moreover, we observed significant increases in BECN1, ATG5, ATG7 and a reduction in SQSTM1/p62 expression relative to irradiated wild-type T cells. We observed similar increases in autophagy gene expression in mice resulting from blockade of CD47 in combination with total body radiation. Pharmacological or siRNA-mediated inhibition of autophagy selectively sensitized CD47-deficient cells to radiation, indicating that enhanced autophagy is necessary for the prosurvival response to CD47 blockade. Moreover, re-expression of CD47 in CD47-deficient T cells sensitized these cells to death by ionizing radiation and reversed the increase in autophagic flux associated with survival. This study indicates that CD47 deficiency confers cell survival through the activation of autophagic flux and identifies CD47 blockade as a pharmacological route to modulate autophagy for protecting tissue from radiation injury.

Hydroxychloroquine Inhibits Autophagy to Potentiate Antiestrogen Responsiveness in ER+ Breast Cancer

Purpose: Estrogen receptor-α (ERα)-targeted therapies including tamoxifen (TAM) or Faslodex (ICI) are used to treat ER+ breast cancers. Up to 50% of tumors will acquire resistance to these interventions. Autophagy has been implicated as a major driver of antiestrogen resistance. We have explored the ability of chloroquine (CQ), which inhibits autophagy, to affect antiestrogen responsiveness. Experimental Design: TAM-resistant MCF7-RR and ICI-resistant/TAM cross-resistant LCC9 ER+ breast cancer cells were injected into mammary fat pads of female athymic mice and treated with TAM and/or ICI in combination with oral low-dose CQ. Results: We show that CQ can increase antiestrogen responsiveness in MCF7-RR and LCC9 cells and tumors, likely through the inhibition of autophagy. However, the combination of ICI+CQ was less effective than CQ alone in vivo, unlike the TAM+CQ combination. Antiestrogen treatment stimulated angiogenesis in tumors but did not prevent CQ effectiveness. The lower efficacy of ICI+CQ was associated with ICI effects on cell-mediated immunity within the tumor microenvironment. The mouse chemokine KC (CXCL1) and IFNγ were differentially regulated by both TAM and ICI treatments, suggesting a possible effect on macrophage development/activity. Consistent with these observations, TAM+CQ treatment increased tumor CD68+ cells infiltration, whereas ICI and ICI+CQ reduced peripheral tumor macrophage content. Moreover, macrophage elimination of breast cancer target cells in vitro was reduced following exposure to ICI. Conclusion: CQ restores antiestrogen sensitivity to resistant tumors. Moreover, the beneficial combination of TAM+CQ suggests a positive outcome for ongoing neoadjuvant clinical trials using this combination for the treatment of ER+ ductal carcinoma in situ lesions. Clin Cancer Res; 20(12); 3222–32. ©2014 AACR.

CD47 in the Tumor Microenvironment Limits Cooperation between Antitumor T-cell Immunity and Radiotherapy

Although significant advances in radiotherapy have increased its effectiveness in many cancer settings, general strategies to widen the therapeutic window between normal tissue toxicity and malignant tumor destruction would still offer great value. CD47 blockade has been found to confer radioprotection to normal tissues while enhancing tumor radiosensitivity. Here, we report that CD47 blockade directly enhances tumor immunosurveillance by CD8(+) T cells. Combining CD47 blockade with irradiation did not affect fibrosarcoma growth in T cell-deficient mice, whereas adoptive transfer of tumor-specific CD8(+) T cells restored combinatorial efficacy. Furthermore, ablation of CD8(+) T cells abolished radiotherapeutic response in immunocompetent syngeneic hosts. CD47 blockade in either target cells or effector cells was sufficient to enhance antigen-dependent CD8(+) CTL-mediated tumor cell killing in vitro. In CD47-deficient syngeneic hosts, engrafted B16 melanomas were 50% more sensitive to irradiation, establishing that CD47 expression in the microenvironment was sufficient to limit tumor radiosensitivity. Mechanistic investigations revealed increased tumor infiltration by cytotoxic CD8(+) T cells in a CD47-deficient microenvironment, with an associated increase in T cell-dependent intratumoral expression of granzyme B. Correspondingly, an inverse correlation between CD8(+) T-cell infiltration and CD47 expression was observed in human melanomas. Our findings establish that blocking CD47 in the context of radiotherapy enhances antitumor immunity by directly stimulating CD8(+) cytotoxic T cells, with the potential to increase curative responses.

Blockade of CD47 increases survival of mice exposed to lethal total body irradiation

Accidental or therapeutic total body exposure to ionizing radiation has profound pathophysiological consequences including acute radiation syndrome. Currently only investigational drugs are available in case of radiological or nuclear accidents or terrorism. Lack of selective radioprotectants for normal tissues also limits the therapeutic doses that can be delivered to treat cancers. CD47 is a receptor for the secreted protein thrombospondin-1. Blockade of thrombospondin-1 or CD47 provides local radioprotection of soft tissues and bone marrow. We now report that suppression of CD47 using an antisense morpholino increases survival of mice exposed to lethal total body irradiation. Increased survival is associated with increased peripheral circulating blood cell counts and increased proliferative capacity of bone marrow derived cells. Moreover, CD47 blockade decreased cell death while inducing a protective autophagy response in radiosensitive gastrointestinal tissues. Thus, CD47 is a new target for radiomitigation that prevents both hematopoietic and gastrointestinal radiation syndromes.

Mitochondria directly donate their membrane to form autophagosomes during a novel mechanism of parkin-associated mitophagy

BackgroundAutophagy (macroautophagy), a cellular process of “self-eating”, segregates damaged/aged organelles into vesicles, fuses with lysosomes, and enables recycling of the digested materials. The precise origin(s) of the autophagosome membrane is unclear and remains a critical but unanswered question. Endoplasmic reticulum, mitochondria, Golgi complex, and the plasma membrane have been proposed as the source of autophagosomal membranes.FindingsUsing electron microscopy, immunogold labeling techniques, confocal microscopy, and flow cytometry we show that mitochondria can directly donate their membrane material to form autophagosomes. We expand upon earlier studies to show that mitochondria donate their membranes to form autophagosomes during basal and drug-induced autophagy. Moreover, electron microscopy and immunogold labeling studies show the first physical evidence of mitochondria forming continuous structures with LC3-labeled autophagosomes. The mitochondria forming these structures also stain positive for parkin, indicating that these mitochondrial-formed autophagosomes represent a novel mechanism of parkin-associated mitophagy.ConclusionsWith the on-going debate regarding autophagosomal membrane origin, this report demonstrates that mitochondria can donate membrane materials to form autophagosomes. These structures may also represent a novel form of mitophagy where the mitochondria contribute to the formation of autophagosomes. This novel form of parkin-associated mitophagy may be a more efficient bio-energetic process compared with de novo biosynthesis of a new membrane, particularly if the membrane is obtained, at least partly, from the organelle being targeted for later degradation in the mature autolysosome.

Angiotensin peptides and lung cancer.

Lung cancer is a leading cause of death in both men and women, with over 1,000,000 new cases diagnosed worldwide annually and a 5-year survival rate of only 14%, a figure that has improved little in the past thirty years. This poor prognosis suggests a need for novel approaches for the treatment and prevention of lung cancer. The renin-angiotensin system is an established, primary regulator of blood pressure, homeostasis, and natriuresis; however, compelling evidence indicates that the angiotensin peptides also play a role in cell proliferation and inflammation. Angiotensin II is a vasoconstrictor, a mitogen, and an angiogenic factor, while angiotensin-(1-7) has vasodilator, anti-proliferative, and anti-angiogenic properties. This review focuses on studies examining the renin-angiotensin system in pulmonary cancers and whether clinical intervention of this pathway may serve as an effective chemotherapeutic and/or chemopreventive modality for lung cancer.