Eco-evolution of cancer resistance.

For the past 60 years, the goal of conventional cancer therapies has been the eradication of every cancer cell. To this end, patients are subjected to the highest possible doses of radiation and chemotherapy as well as radical surgeries. In the rare case in which eradication was possible, clinicians achieved long-term control of the disease. For the most part, however, upfront eradication is not possible, and despite intensive and very toxic therapies, the patient dies of the disease or of complications of therapy. As our understanding about the role of tumor microenvironment in tumor progression and drug resistance improves, we are realizing that the paradigm of killing all cancer cells may be flawed.

Cancer Therapy Targeting the HER2-PI3K Pathway: Potential Impact on the Heart.

The HER2-PI3K pathway is the one of the most mutated pathways in cancer. Several drugs targeting the major kinases of this pathway have been approved by the Food and Drug Administration and many are being tested in clinical trials for the treatment of various cancers. However, the HER2-PI3K pathway is also pivotal for maintaining the physiological function of the heart, especially in the presence of cardiac stress. Clinical studies have shown that in patients treated with doxorubicin concurrently with Trastuzumab, a monoclonal antibody that blocks the HER2 receptor, the New York Heart Association class III/IV heart failure was significantly increased compared to those who were treated with doxorubicin alone (16 vs. 3%). Studies in transgenic mice have also shown that other key kinases of this pathway, such as PI3Kα, PDK1, Akt, and mTOR, are important for protecting the heart from ischemia-reperfusion and aortic stenosis induced cardiac dysfunction. Studies, however, have also shown that inhibition of PI3Kγ improve cardiac function of a failing heart. In addition, results from transgenic mouse models are not always consistent with the outcome of the pharmacological inhibition of this pathway. Here, we will review these findings and discuss how we can address the cardiac side-effects caused by inhibition of this important pathway in both cancer and cardiac biology.

VEGF, PF4 and PDGF are elevated in platelets of colorectal cancer patients.

Platelets sequester angiogenesis regulatory proteins which suggests an avenue for developing biomarkers to monitor disease. We describe a comparison of angiogenesis regulatory proteins found in platelets of colorectal cancer patients and normal controls. Platelet and plasma content of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF), platelet factor 4 (PF4), thrombospondin-1 (TSP-1) and endostatin in 35 patients with colon cancer were compared with 84 age-matched healthy controls using ELISAs. We standardized the platelet preparation procedure, introduced process controls and normalized the respective protein levels to platelet numbers using an actin ELISA. Statistically significant differences were found in the median levels of VEGF, PF4 and PDGF in platelets of patients with cancer compared to healthy individuals. Platelet concentrations in cancer patients versus controls were: VEGF 1.3 versus 0.6 pg/10(6), PF4 18.5 versus 9.4 ng/10(6), and PDGF 34.1 versus 21.0 pg/10(6). Multivariable logistic regression analysis indicated that PDGF, PF4 and VEGF were independent predictors of colorectal carcinoma and as a set provided statistically significant discrimination (area under the curve = 0.893, P < .0001). No significant differences were detected for bFGF, endostatin, or TSP-1. Reference Change Value analysis determined that the differences seen were not clinically significant. Plasma levels yielded no correlations.

Isolation and proteomic analysis of platelets by SELDI-TOF MS.

Many growth factors, leukotrines, and biological ligands are not circulating free in plasma or serum, except in the case of late or disseminated disease. During early tumor growth and angiogenesis, platelets actively and selectively sequester regulators of angiogenesis and, as such, the platelet protein content can be used as a marker of early tumor growth or angiogenesis. With the recent increase in the clinical use of biologic modifiers in cancer and chronic disease therapy, the search for markers of early disease, therapeutic response, and/or recurrence has suggested that analysis of platelet proteins may be more relevant and accurate. We provide a guideline for the proteomic analysis of platelet proteome, placing specific emphasis on angiogenesis regulators, even though other platelet proteins may serve as markers of disease in the future. The analysis of serum/plasma has been fraught with difficulties because of the extraordinarily large number of proteins and because some of the proteins are contained in extraordinarily large amounts, masking the less abundant proteins. Thus, platelets may provide a much more biologically relevant analyte for biomarker discovery.

Normal ranges of angiogenesis regulatory proteins in human platelets.

Platelets sequester angiogenesis regulatory proteins early in tumor growth, which suggests a new avenue for monitoring disease. To date, there are no clinically relevant reference ranges for markers of early angiogenesis. We introduce a new ELISA-based method for accurate and reproducible measurement of vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), platelet factor 4 (PF4), thrombospondin-1 (TSP-1), fibroblast growth factor, basic (bFGF), and endostatin in platelets. To facilitate clinical applicability, the platelet levels in isolated samples were determined utilizing a new actin ELISA method. Platelets from healthy donors at single and repetitive time points were used for the assessment of normal ranges of these proteins. The physiological levels in platelets were: VEGF (0.74 +/- 0.37 pg/10(6) platelets); PDGF (23 +/- 6 pg/10(6)); PF4 (12 +/- 5 ng/10(6)); TSP-1 (31 +/- 12 ng/10(6)); bFGF (0.44 +/- 0.15 pg/10(6)); and endostatin (5.6 +/- 3.0 pg/10(6)). There was an excellent correlation (R(2) = 0.7) between the platelet levels calculated with the actin ELISA and complete blood count. The levels of the platelets were higher than those in platelet-poor plasma by factors of: VEGF (215-fold); PDGF (914-fold); PF-4 (516-fold); TSP-1 (813-fold); and bFGF (17-fold). The endostatin levels were nearly equivalent. The biovariability of the platelet proteins in eight healthy subjects over a 5-week period was found to be minimal. We describe accurate and direct measurements of the concentrations of VEGF, bFGF, PDGF, TSP-1, endostatin, and PF4 in platelets of healthy human subjects. In contrast to the highly variable levels in plasma and serum, the platelet-derived measurements were accurate and reproducible with minimal biovariability.

Pharmacological treatment of a diffuse arteriovenous malformation of the upper extremity in a child.

A young girl with an arteriovenous malformation involving the right upper extremity developed rapidly progressive bony destruction that did not respond to embolization. Treatment with marimastat, starting at 3 years of age, resulted in rapid resolution of pain and gradual healing of bony destruction, associated with regression of the intraosseous arteriovenous shunts. New arteriovenous shunts with bony destruction developed over the years and responded to an increase in the dose of marimastat. Interruption of therapy resulted in recurrence of pain and formation of new lesions. The patient has been treated in this way for 12 years with no adverse effects from the drug.

Megakaryocyte-derived microparticles: direct visualization and distinction from platelet-derived microparticles.

Platelet microparticles are a normal constituent of circulating blood. Several studies have demonstrated positive correlations between thrombotic states and platelet microparticle levels. Yet little is known about the processes by which platelet microparticles are generated in vivo. We now characterize microparticles derived directly from megakaryocytes. Video microscopy of live mouse megakaryocytes demonstrated that microparticles form as submicron beads along the lengths of slender, unbranched micropodia. These microparticles are CD41(+), CD42b(+), and express surface phosphatidylserine. Megakaryocyte microparticle generation is resistant to inhibition of microtubule assembly, which is critical to platelet formation, and augmented by inhibition of actin polymerization. To determine whether circulating microparticles are derived primarily from activated platelets or megakaryocytes, we identified markers that distinguish between these 2 populations. CD62P and LAMP-1 were found only on mouse microparticles from activated platelets. In contrast, full-length filamin A was found in megakaryocyte-derived microparticles, but not microparticles from activated platelets. Circulating microparticles isolated from mice were CD62P(-), LAMP-1(-) and expressed full-length filamin A, indicating a megakaryocytic origin. Similarly, circulating microparticles isolated from healthy volunteers were CD62P(-) and expressed full-length filamin A. Cultured human megakaryocytes elaborated microparticles that were CD41(+), CD42b(+), and express surface phosphatidylserine. These results indicate that direct production by megakaryocytes represents a physiologic means to generate circulating platelet microparticles.

Platelet-associated PF-4 as a biomarker of early tumor growth.

Early tumor detection and intervention are important determinants of survival in patients with cancer. We have recently reported that the "platelet angiogenesis proteome" may be used to detect microscopic tumors in mice. We now present evidence that changes in platelet-associated platelet factor-4 (PF-4) detect malignant growth across a spectrum of human cancers in mice. A deregulated expression of an 8206-Da protein was observed by surfaceenhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-ToF MS) proteomic comparison of platelets from normal and tumor-bearing mice. The differentially expressed protein was identified as PF-4 by tandem mass spectrometry and ProteinChip immunoassay using anti-PF-4 antibody. The platelet-associated PF-4 appeared to be up-regulated in early growth of human liposarcoma, mammary adenocarcinoma, and osteosarcoma. A 120-day follow-up study of liposarcoma revealed a sustained 2-fold or higher increase of platelet-associated PF-4 at 19, 30, and 120 days. In contrast, only an insignificant change of PF-4 was observed in the plasma of mice bearing the different human tumor xenografts, and throughout the 120 days of the liposarcoma study. We conclude that platelet-associated PF-4, but not its plasma counterpart, may represent a potential biomarker of early tumor presence.

Angiogenesis is regulated by a novel mechanism: pro- and antiangiogenic proteins are organized into separate platelet alpha granules and differentially released.

Platelets, in addition to their function in hemostasis, play an important role in wound healing and tumor growth. Because platelets contain angiogenesis stimulators and inhibitors, the mechanisms by which platelets regulate angiogenesis remain unclear. As platelets adhere to activated endothelium, their action can enhance or inhibit local angiogenesis. We therefore suspected a higher organization of angiogenesis regulators in platelets. Using double immunofluorescence and immunoelectron microscopy, we show that pro- and antiangiogenic proteins are separated in distinct subpopulations of alpha-granules in platelets and megakaryocytes. Double immunofluorescence labeling of vascular endothelial growth factor (VEGF) (an angiogenesis stimulator) and endostatin (an angiogenesis inhibitor), or for thrombospondin-1 and basic fibroblast growth factor, confirms the segregation of stimulators and inhibitors into separate and distinct alpha-granules. These observations motivated the hypothesis that distinct populations of alpha-granules could undergo selective release. The treatment of human platelets with a selective PAR4 agonist (AYPGKF-NH(2)) resulted in release of endostatin-containing granules, but not VEGF-containing granules, whereas the selective PAR1 agonist (TFLLR-NH(2)) liberated VEGF, but not endostatin-containing granules. In conclusion, the separate packaging of angiogenesis regulators into pharmacologically and morphologically distinct populations of alpha-granules in megakaryocytes and platelets may provide a mechanism by which platelets can locally stimulate or inhibit angiogenesis.