Adam S. Curatolo

Bone marrow is a reservoir for proangiogenic myelomonocytic cells but not endothelial cells in spontaneous tumors

The hypothesis that bone marrow-derived, circulating endothelial cells incorporate into tumor blood vessels is unresolved. We have measured the numbers of bone marrow-derived versus resident endothelial cells in spontaneous prostate cancers during different stages of tumor progression and in age-matched normal prostates. Bone marrow-derived endothelial cells were rare in dysplasia and in well differentiated cancers representing between 0 and 0.04% of the total tumor mass. Instead, approximately 99% of all tumor-associated bone marrow-derived cells were CD45(+) hematopoietic cells, including GR-1(+), F4-80(+), and CD11b(+) myeloid cells. Similar to peripheral blood mononuclear cells, these tumor-associated myeloid cells expressed matrix metalloproteinases (MMPs), consistent with their proposed catalytic role during tumor angiogenesis. Furthermore, freshly isolated CD11b(+) cells stimulated tumor endothelial cell cord formation by 10-fold in an in vitro angiogenesis assay. The bone marrow is, therefore, a reservoir for cells that augment tumor angiogenesis, but the tumor endothelium is derived primarily from the local environment.

Hypercholesterolemia induces angiogenesis and accelerates growth of breast tumors in vivo.

Obesity and metabolic syndrome are linked to an increased prevalence of breast cancer among postmenopausal women. A common feature of obesity, metabolic syndrome, and a Western diet rich in saturated fat is a high level of circulating cholesterol. Epidemiological reports investigating the relationship between high circulating cholesterol levels, cholesterol-lowering drugs, and breast cancer are conflicting. Here, we modeled this complex condition in a well-controlled, preclinical animal model using innovative isocaloric diets. Female severe combined immunodeficient mice were fed a low-fat/no-cholesterol diet and then randomized to four isocaloric diet groups: low-fat/no-cholesterol diet, with or without ezetimibe (cholesterol-lowering drug), and high-fat/high-cholesterol diet, with or without ezetimibe. Mice were implanted orthotopically with MDA-MB-231 cells. Breast tumors from animals fed the high-fat/high-cholesterol diet exhibited the fastest progression. Significant differences in serum cholesterol level between groups were achieved and maintained throughout the study; however, no differences were observed in intratumoral cholesterol levels. To determine the mechanism of cholesterol-induced tumor progression, we analyzed tumor proliferation, apoptosis, and angiogenesis and found a significantly greater percentage of proliferating cells from mice fed the high-fat/high-cholesterol diet. Tumors from hypercholesterolemic animals displayed significantly less apoptosis compared with the other groups. Tumors from high-fat/high-cholesterol mice had significantly higher microvessel density compared with tumors from the other groups. These results demonstrate that hypercholesterolemia induces angiogenesis and accelerates breast tumor growth in vivo.

Urinary MMP-2 and MMP-9 predict the presence of ovarian cancer in women with normal CA125 levels

OBJECTIVE To determine whether urinary matrix metalloproteinases (MMPs) predict the presence of ovarian cancer in patients with CA125 levels below the normal threshold of 35U/mL, a critical group of patients for whom no ovarian cancer biomarker is currently available. To determine whether these noninvasive biomarkers provide clinically useful information in the general ovarian cancer patient population as well. METHODS ELISA analyses and substrate gel electrophoresis detected the levels and activity of urinary MMP-2, MMP-9, MMP-9/neutrophil gelatinase-associated lipocalin (NGAL) complex, and MMP-9 dimer in all ovarian cancer patients (n=97), those with CA125 <35U/mL (n=26) and controls (n=81). RESULTS In patients with CA125 <35U/mL, receiver-operating characteristic (ROC) area under curve (AUC) analysis demonstrated that either urinary MMP-2 or MMP-9 or NGAL significantly discriminated between controls and ovarian cancer patients with normal CA125. Multivariate logistic regression revealed that the combination of urinary MMP-2 and MMP-9 provided the best diagnostic accuracy when multiplexed. When further multiplexed with age, the diagnostic accuracy of these biomarkers increased to a significant AUC of 0.820. These findings were consistent among the general ovarian cancer population studied as well, where the combination of urinary MMP-2 and MMP-9 multiplexed with age resulted in a highly significant AUC of 0.881. Pearson chi-square analysis revealed that higher urinary levels of either MMP-2 or MMP-9 were strongly associated with the increasing percentage of women with ovarian cancer independent of CA125 levels. CONCLUSION This study demonstrates the potential utility of urinary MMP-2 and MMP-9 to differentiate between ovarian cancer patients with normal CA125 levels and controls and suggests that urinary MMP-2 and MMP-9 may be a clinically useful aid in the diagnosis of advanced or recurrent ovarian cancer.

Novel non-invasive biomarkers that distinguish between benign prostate hyperplasia and prostate cancer

BackgroundThe objective of this study was to discover and to validate novel noninvasive biomarkers that distinguish between benign prostate hyperplasia (BPH) and localized prostate cancer (PCa), thereby helping to solve the diagnostic dilemma confronting clinicians who treat these patients.MethodsQuantitative iTRAQ LC/LC/MS/MS analysis was used to identify proteins that are differentially expressed in the urine of men with BPH compared with those who have localized PCa. These proteins were validated in 173 urine samples from patients diagnosed with BPH (N = 83) and PCa (N = 90). Multivariate logistic regression analysis was used to identify the predictive biomarkers.ResultsThree proteins, β2M, PGA3, and MUC3 were identified by iTRAQ and validated by immunoblot analyses. Univariate analysis demonstrated significant elevations in urinary β2M (P < 0.001), PGA3 (P = 0.006), and MUC3 (P = 0.018) levels found in the urine of PCa patients. Multivariate logistic regression analysis revealed AUC values ranging from 0.618 for MUC3 (P = 0.009), 0.625 for PGA3 (P < 0.008), and 0.668 for β2M (P < 0.001). The combination of all three demonstrated an AUC of 0.710 (95% CI: 0.631 – 0.788, P < 0.001); diagnostic accuracy improved even more when these data were combined with PSA categories (AUC = 0.812, (95% CI: 0.740 – 0.885, P < 0.001).ConclusionsUrinary β2M, PGA3, and MUC3, when analyzed alone or when multiplexed with clinically defined categories of PSA, may be clinically useful in noninvasively resolving the dilemma of effectively discriminating between BPH and localized PCa.

Cooperative benefit for the combination of rapamycin and imatinib in tuberous sclerosis complex neoplasia

Tuberous sclerosis (TS) is a common autosomal-dominant disorder characterized by tumors of the skin, lung, brain, and kidneys. Monotherapy with rapamycin however resulted in partial regression of tumors, implying the involvement of additional pathways. We have previously implicated platelet-derived growth factor-BB in TS-related tumorigenesis, thus providing a rationale for a combination of mTOR/PDGF blockade using rapamycin and imatinib. Here, we test this combination using a well-established preclinical model of cutaneous tumorigenesis in TS, tsc2ang1 cells derived from a skin tumor from a mouse heterozygous for tsc2. Treatment of tsc2ang1 cells with a combination of rapamycin and imatinib led to an inhibition of proliferation compared with either vehicle treatment or treatment with rapamycin or imatinib monotherapy. Combination therapy also led to a decrease in Akt activation. Potent in vivo activity in animal experiments by combination therapy was noted, without toxicity to the animals. Our findings provide a rationale for the combined use of rapamycin and imatinib, both FDA approved drugs, for the treatment of TS.

Urine vascular biomarkers in Sturge–Weber syndrome

Sturge–Weber syndrome (SWS) consists of a capillary-venous vascular malformation of the brain, skin and eye. Urine vascular biomarkers have been demonstrated to be abnormal in other vascular anomalies and to correlate with clinical severity and progression. The current study investigated the use of urinary matrix metalloproteinase (MMP)-2, MMP-9, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) levels to non-invasively monitor the progression of SWS. Fifty-four urine samples were collected from patients seen at the Hunter Nelson Sturge–Weber Center at Kennedy Krieger Institute. Urine was analyzed for MMP-2, MMP-9, VEGF and bFGF levels and correlated with clinical outcome at the time of urine collection (n = 48) and 1 year following urine collection (n = 22). Analysis revealed that MMP-2 (p = 0.033) and MMP-9 (p = 0.010) were significantly more likely to be present in the urine of SWS subjects compared to controls and that bFGF was significantly more likely to be present at abnormal levels (p = 0.005). MMP-2 correlated with a more severe clinical score at the time of urine collection, while both MMP-2 and MMP-9 levels correlated with greater disease severity at time of collection. bFGF levels correlated with improved clinical score 1 year after urine collection. These results suggest that MMP-2 and MMP-9 levels may be useful in assessing SWS progression, as well as indicating which patients might benefit from more aggressive treatment, while bFGF levels may be useful in judging the efficacy of neurologic treatment in SWS.

Age-Dependent Loss of MMP-3 in Hutchinson–Gilford Progeria Syndrome

Hutchinson-Gilford progeria syndrome (HGPS) is a rare, progressive segmental premature aging disease that includes scleroderma-like skin, progressive joint contracture, and atherosclerosis. Affected individuals die prematurely of heart attacks or strokes. Extracellular matrix dysregulation is implicated as a factor in disease progression. We analyzed messenger RNA and protein levels for matrix metalloproteinases (MMPs)-2,-3, and -9 in HGPS primary human dermal fibroblasts using real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and gelatin zymography. MMP-3 messenger RNA and protein levels decreased significantly with increasing donor age in HGPS fibroblasts but not in controls. MMP-2 messenger RNA also showed a donor age-dependent decrease in HGPS fibroblasts, but levels of secreted protein were unchanged. MMP-9 was similar in HGPS and control cultures. The decreased MMP-3 may represent a shift in the inherent extracellular matrix-degrading proteolytic balance in favor of matrix deposition in HGPS. This metalloproteinase has the potential to serve as a biomarker of therapeutic efficacy when assessing treatments for HGPS.

Identification of novel non-invasive biomarkers of urinary chronic pelvic pain syndrome: findings from the Multidisciplinary Approach to the Study of Chronic Pelvic Pain (MAPP) Research Network

To examine a series of candidate markers for urological chronic pelvic pain syndrome (UCPPS), selected based on their proposed involvement in underlying biological processes so as to provide new insights into pathophysiology and suggest targets for expanded clinical and mechanistic studies.

Prenatal urinary matrix metalloproteinase profiling as a potential diagnostic tool in fetal obstructive uropathy

BACKGROUND/PURPOSE The diagnostic evaluation, patient stratification, and prenatal counseling for congenital obstructive uropathy remain sub-optimal. Matrix metalloproteinase (MMP) expression profiles are emerging as a valuable diagnostic tool in assorted disease processes. We sought to determine whether congenital obstructive uropathy impacts MMP expression in fetal urine. METHODS Fetal lambs (n = 25) were divided in two groups: group I (n = 12) underwent a sham operation and group II (n = 13) underwent creation of a complete urinary tract obstruction. Gelatin zymography panels for 4 MMP species were performed on fetal urine in both groups at comparable times post-operatively. Statistical analysis was by the Fishers exact test (P < .05). RESULTS Overall fetal survival was 80% (20/25). A variety of significant differences in MMP expression between the two groups were identified. The following profiles were present only in obstructed animals: any MMP other than MMP-2 (P = .029), including any MMP other than 63 kDa and 65 kDa (P = .009); 2 or more MMPs excluding MMP-2s (0.029); and 3 or more MMPs (P = .029). CONCLUSIONS Limited matrix metalloproteinase expression is present in the urine of normal ovine fetuses. Fetal obstructive uropathy impacts urinary MMP expression in various distinguishable patterns. Prenatal urinary MMP profiling may become a practical and valuable diagnostic tool in the evaluation of congenital obstructive uropathy.

Tris DBA palladium is highly effective against growth and metastasis of pancreatic cancer in an orthotopic model

Pancreatic carcinoma ranks among the most lethal of human cancers. Besides late detection, other factors contribute to its lethality, including a high degree of chemoresistance, invasion, and distant metastases. Currently, the mainstay of therapy involves resection of local disease in a minority of patients (Whipple procedure) and systemic gemcitabine. While systemic chemotherapy has some benefit, even with optimal treatment, the five year survival after diagnosis is dismal. Thus, treatment of pancreatic carcinoma remains a tremendous unmet need. The organometallic compound tris DBA palladium is a potent inhibitor of N-myristoyltransferase 1 (NMT1), an enzyme that catalyzes the transfer of myristate to protein substrates. This compound is highly effective in vivo against murine models of melanoma with both mutant and wild type b-RAF genotypes. Based upon the signaling similarities between melanoma and pancreatic carcinoma, we evaluated the efficacy of tris DBA palladium in vitro and in vivo against pancreatic carcinoma. We found that tris DBA palladium decreased proliferation and colony formation of pancreatic cancer cells in vitro. In an orthotopic mouse model, tris DBA palladium was highly active in inhibiting growth, ascites production, and distant metastases in vivo. Furthermore, tris DBA palladium impaired chemotaxis and inhibited cilia formation in Pan02 cells in a NMT1-dependent manner. We propose that NMT1 is a novel regulator of cilia formation and tris DBA palladium a novel inhibitor of cilia formation and metastasis in pancreatic cancer. Thus, further evaluation of tris DBA palladium for the treatment of pancreatic cancer is warranted.