Madhuchhanda Roy

Brain metastases in melanoma: roles of neurotrophins.

Brain metastasis, which occurs in 20% to 40% of all cancer patients, is an important cause of neoplastic morbidity and mortality. Successful invasion into the brain by tumor cells must include attachment to microvessel endothelial cells, penetration through the blood-brain barrier, and, of relevance, a response to brain survival and growth factors. Neurotrophins (NTs) are important in brain-invasive steps. Human melanoma cell lines express low-affinity NT receptor p75NTR in relation to their brain-metastatic propensity with their invasive properties being regulated by NGF, or nerve growth factor, the prototypic NT. They also express functional TrkC, the putative receptor for the invasion-promoting NT-3. In brain-metastatic melanoma cells, NTs promote invasion by enhancing the production of extracellular matrix (ECM)-degradative enzymes such as heparanase, an enzyme capable of locally destroying both ECM and the basement membrane of the blood-brain barrier. Heparanase is an endo-beta-d-glucuronidase that cleaves heparan sulfate (HS) chains of ECM HS proteoglycans, and it is a unique metastatic determinant because it is the dominant mammalian HS degradative enzyme. Brain-metastatic melanoma cells also produce autocrine/paracrine factors that influence their growth, invasion, and survival in the brain. Synthesis of these factors may serve to regulate NT production by brain cells adjacent to the neoplastic invasion front, such as astrocytes. Increased NT levels have been observed in tumor-adjacent tissues at the invasion front of human brain melanoma. Additionally, astrocytes may contribute to the brain-metastatic specificity of melanoma cells by producing NT-regulated heparanase. Trophic, autocrine, and paracrine growth factors may therefore determine whether metastatic cells can successfully invade, colonize, and grow in the CNS.

Inhibition of heparanase activity and heparanase‐induced angiogenesis by suramin analogues

Heparanase, a heparan sulfate‐specific endo‐β‐D‐glucuronidase, plays an important role in tumor cell metastasis through the degradation of extracellular matrix heparan sulfate proteoglycans (ECM HSPG). Heparanase activity correlates with the metastatic propensity of tumor cells. Suramin, a polysulfonated naphthylurea, is an inhibitor of heparanase with suramin analogues shown to possess antiangiogenic and antiproliferative properties. We investigated the effects of selected suramin analogues (NF 127, NF 145 and NF 171) on heparanase activity and heparanase‐driven angiogenesis. Studies of the ability of cellular extracts and purified heparanase from human, highly invasive and brain‐metastatic melanoma (70W) cells revealed that heparanase expressed by these cells was effectively inhibited by suramin analogues in a dose‐dependent manner. These analogues possessed more potent heparanase inhibitory activities than suramin: The concentrations required for 50% heparanase inhibition (IC50) were 20–30 μM, or at least 2 times lower than that for suramin. One hundred percent inhibition was observed at concentrations of 100 μM and higher. Of relevance, these compounds significantly decreased (i) the invasive capacity of human 70W cells by chemoinvasion assays performed with filters coated with purified HSPG or Matrigel™, and (ii) blood vessel formation by in vivo angiogenic assays, thus linking their antiangiogenic properties with impedance of heparanase‐induced angiogenesis. Specifically, inhibition of invasion by NF 127, NF 145 and NF 171 was found at 10 μM concentrations of compounds with a significant decrease of invasive values at concentrations as low as 1.5 μM. In addition, NF 127, NF 145 and NF 171 promoted nearly complete inhibition of heparanase‐induced angiogenesis at values ranging from 236 μM (for NF 145) to 362 μM (for NF 127). These results further emphasize the importance of heparanase in invasive and angiogenic mechanisms and the potential clinical application of heparanase inhibitors such as suramin analogues in cancers and angiogenesis‐dependent diseases.

Cell surface heparan sulfate released by heparanase promotes melanoma cell migration and angiogenesis.

Heparan sulfate (HS) proteoglycans are essential components of the cell‐surface and extracellular matrix (ECM) which provide structural integrity and act as storage depots for growth factors and chemokines, through their HS side chains. Heparanase (HPSE) is the only mammalian endoglycosidase known that cleaves HS, thus contributing to matrix degradation and cell invasion. The enzyme acts as an endo‐β‐D‐glucuronidase resulting in HS fragments of discrete molecular weight size. Cell‐surface HS is known to inhibit or stimulate tumorigenesis depending upon size and composition. We hypothesized that HPSE contributes to melanoma metastasis by generating bioactive HS from the cell‐surface to facilitate biological activities of tumor cells as well as tumor microenvironment. We removed cell‐surface HS from melanoma (B16B15b) by HPSE treatment and resulting fragments were isolated. Purified cell‐surface HS stimulated in vitro B16B15b cell migration but not proliferation, and importantly, enhanced in vivo angiogenesis. Furthermore, melanoma cell‐surface HS did not affect in vitro endothelioma cell (b.End3) migration. Our results provide direct evidence that, in addition to remodeling ECM and releasing growth factors and chemokines, HPSE contributes to aggressive phenotype of melanoma by releasing bioactive cell‐surface HS fragments which can stimulate melanoma cell migration in vitro and angiogenesis in vivo. J. Cell. Biochem. 106: 200–209, 2009.

Brain-metastatic melanoma: a neurotrophic perspective

The brain is a unique microenvironment enclosed by the skull and maintaining a highly regulated vascular transport barrier. To metastasize to the brain, malignant tumor cells must attach to microvessel endothelial cells, invade the blood-brain barrier (BBB), and respond to brain survival and growth factors. Neurotrophins (NT) are important in brain invasion because they stimulate this process. In brain-metastatic melanoma cells, NT can promote invasion by enhancing the production of extracellular matrixdegradative enzymes such as heparanase, an enzyme capable of locally destroying both the extracellular matrix and the basement membrane of the BBB. We have examined human and murine melanoma cell lines exhibiting varying abilities to form brain metastases, and have found that they express low-affinity neurotrophin receptor p75NTR in relation to their brain-metastatic potentials. They do not, however, express trkA, the gene encoding the tyrosine kinase receptor TrkA, the high-affinity receptor for nerve growth factor (NGF), the prototypic NT. Presence of functional TrkC, the putative receptor for the invasion-promoting neurotrophin NT-3, was also expressed in these cells. Brain-metastatic melanoma cells can also produce autocrine factors and inhibitors that influence their growth, invasion, and survival in the brain. Synthesis of these factors may influence NT production by brain cells adjacent to the neoplastic invasion front, such as oligodendrocytes and astrocytes. In brain biopsies, we observed increased amounts of NGF and NT-3 in tumor-adjacent tissues at the invasion front of human melanoma tumors. Additionally, we found that astrocytes contribute to the brain-metastatic specificity of melanoma cells by producing NT-regulated heparanase. Trophic, autocrine, and paracrine growth factors may therefore determine whether metastatic cells can successfully invade, colonize, and grow in the central nervous system (CNS).

Vascular endothelial‐cadherin stimulates syndecan‐1‐coupled insulin‐like growth factor‐1 receptor and cross‐talk between αVβ3 integrin and vascular endothelial growth factor receptor 2 at the onset of endothelial cell dissemination during angiogenesis

Vascular endothelial growth factor (VEGF)‐stimulated angiogenesis depends on a cross‐talk mechanism involving VEGF receptor 2 (VEGFR2), vascular endothelial (VE)‐cadherin and the αVβ3 integrin. Because we have shown that αVβ3 integrin activation is dependent on its incorporation, along with the insulin‐like growth factor‐1 receptor (IGF1R) kinase, into a ternary receptor complex organized by the matrix receptor syndecan‐1 (Sdc1), we questioned the role of this core complex in VEGF‐stimulated angiogenesis. We find that the Sdc1‐coupled ternary receptor complex is required for VEGF signalling and for stimulation of vascular endothelial cell migration by vascular endothelial cadherin (VE‐cadherin) engagement. VE‐cadherin binding to Fc/VE‐cadherin extracellular domain chimera activates Sdc1‐coupled IGF1R and αvβ3 integrin; this depends on VEGFR2 and c‐Src activated by the cadherin. Blocking homotypic VE‐cadherin engagement disrupts VEGF‐stimulated cell migration, which is restored by clustering the cadherin in the absence of cell–cell adhesion. This cadherin‐dependent stimulation requires VEGFR2 and IGF1R and is blocked by synstatin (SSTN)92–119, a peptide that competitively disrupts the Sdc1‐coupled ternary complex and prevents the αVβ3 integrin activation required for VEGFR2 activation. VEGFR2‐stimulated angiogenesis in the mouse aortic ring explant assay is disrupted by SSTN, although only early in the process, suggesting that IGF1R coupling to Sdc1 and αVβ3 integrin comprises a core activation mechanism activated by VE‐cadherin that is necessary for VEGFR2 and integrin activation in the initial stages of endothelial cell dissemination during angiogenesis.

Current Understanding and Management of Medullary Thyroid Cancer

Medullary thyroid cancer (MTC) typically accounts for 3%-4% of all thyroid cancers. Although the majority of MTCs are sporadic, 20% of cases are hereditary. Hereditary MTC can be found in multiple endocrine neoplasia 2A or 2B or as part of familial MTC based on a specific germline mutation in the RET proto-oncogene. This article discusses the current approaches available for the diagnosis, evaluation, and management of patients and their family members with suspected MTC. The disease is predominantly managed surgically and typically requires a total thyroidectomy and lymph node dissection. A review of recent guidelines on the extent and timing of surgical excision is discussed. There are not very many effective systemic treatment options for MTC, but several emerging therapeutic targets have promise.

Is DVT prophylaxis necessary for thyroidectomy and parathyroidectomy

BACKGROUND Recent guidelines suggest pharmacologic deep vein thrombosis (DVT) prophylaxis in all patients undergoing major surgical procedures to minimize the risk of postoperative DVT and pulmonary embolism (PE). Pharmacologic DVT prophylaxis perioperatively might increase the risk of bleeding complications. Our goal was to study the risk/benefit ratio of DVT prophylaxis in patients who undergo thyroidectomy and parathyroidectomy. METHODS A review of the ACS NSQIP Database from 2005 to 2007 was performed. The incidence of DVT/PE complications in a cohort of 347,862 patients was compared with the 16,022 patients who underwent a thyroidectomy or parathyroidectomy. We identified risk factors for DVT/PE and developed a surrogate variable to determine the risk for postoperative bleeding. RESULTS The risk of DVT/PE complication in the thyroidectomy and parathyroidectomy patients (0.16) was 6 fold less than the entire cohort (0.96) (P < .001). The estimated risk of bleeding requiring a return to the operating room was 1.58%, which is 10-fold greater than the risk of developing a DVT/PE (P < .001). CONCLUSION Patients who underwent thyroidectomy and parathyroidectomy have a low incidence of developing DVT/PE complications and have a significantly greater risk of developing bleeding complications. Hence, we believe that DVT prophylaxis should be done at the discretion of the surgeon in select high-risk patients only.

Persistent hypertension after adrenalectomy for an aldosterone-producing adenoma: weight as a critical prognostic factor for aldosterone's lasting effect on the cardiac and vascular systems.

BACKGROUND Primary aldosteronism caused by an aldosterone producing adrenal tumor/aldosteronoma (APA), is a potentially curable form of hypertension, via unilateral adrenalectomy. Resolution of hypertension (HTN) is not as prevalent after tumor resection, as are the normalization of aldosterone secretion, hypokalemia, and other metabolic abnormalities. Here, we review the immediate and long-term medical outcomes of laparoscopic adrenalectomy in patients with an APA, and attempt to identify any distinctive sex differences in the management of resistant HTN. METHODS We performed a retrospective review of the prospective adrenal database at the University of Wisconsin between January 2001 and October 2010. Of the 165 adrenalectomies performed, 32 were for the resection of an APA. Patients were grouped according to their postoperative HTN status. Those patients with normal blood pressure (≤120/80 mm Hg) and on no antihypertensive medication (CURE) were compared with those who continued to require medication for blood pressure control (HTN). We evaluated sex, age, body mass index, tumor size, duration of time with high blood pressure, and the differences in systolic and diastolic blood pressure following adrenalectomy. Statistical analysis was performed using Students t-test. Statistical significance was defined as a P value of <0.05. RESULTS We identified 32 patients with an APA based on biochemical and radiographic studies, two patients were excluded, due to missing data. There were 19 males (63%) and 11 (37%) females, with a mean age was 48.3 ± 2.1 y, and mean tumor size was 24 ± 3 mm. Postoperatively, patients required significantly fewer antihypertensive medications (1.5 ± 0.2 versus 3.3 ± 0.3, P < 0.001). Nine patients (31%) had complete resolution of their HTN, requiring no postoperative antihypertensive medication. The only significant difference between the sexes, was a lower body mass index in women (27.6 ± 1.7 versus 33.4 ± 2.1 kg/m(2), P = 0.04). Ninety percent of the cohort had at least a 20 mm Hg decline in their systolic blood pressure postoperatively, placing them in the prehypertensive or normal blood pressure categories. Sixty-six percent of the CURE patients required at least 6 mo for resolution of their HTN. All 20 patients who presented with hypokalemia, had immediate resolution postoperatively and did not require continuance of the preoperative spironolactone or potassium supplementation. CONCLUSIONS Laparoscopic adrenalectomy for aldosterone producing adenoma results in the normalization of, or more readily manageable blood pressure in 90% of patients, within 6 mo. Metabolic disturbances are immediately corrected with tumor resection. Weight is an important contributing factor in resolving HTN.

Dominant-negative CREB inhibits heparanase functionality and melanoma cell invasion

Heparanase (HPSE‐1) is an endo‐β‐d‐glucuronidase involved in the degradation of cell‐surface/extracellular matrix heparan sulfate (HS) in normal and neoplastic tissues. HPSE‐1 represents the first example of purification and cloning of a mammalian HS‐degradative enzyme. Elevated HPSE‐1 levels are known to be associated with metastatic cancers, directly implicating HPSE‐1 in metastatic events. The purpose of this study was to determine the role of cAMP response element‐binding protein (CREB) in modulating HPSE‐1‐mediated effects on human melanoma cell invasion. Highly invasive, brain‐metastatic melanoma cells (70W) were transfected with the dominant‐negative CREB (KCREB) and subsequently analyzed for changes in their HPSE‐1 content, functionality, and cell invasive properties. KCREB‐transfected cells showed a decrease in HPSE‐1 mRNA expression and activity. This correlated with a significantly decreased invasion of these cells through Matrigel™‐coated filters. Furthermore, adenoviral vectors containing the full‐length human HPSE‐1 cDNA in sense orientation (Ad‐S/hep) were constructed to investigate CREB effects on HPSE‐1. Restoration of HPSE‐1 expression and functionality following Ad‐S/hep infection of KCREB‐transfected 70W cells recovered melanoma cell invasiveness. These results demonstrate that KCREB inhibits HPSE‐1 and suggest that one of the roles CREB plays in the acquisition of melanoma cells metastatic phenotype is affecting HPSE‐1 activity.

Epidermal growth factor regulates NIKS keratinocyte proliferation through Notch signaling

BACKGROUND Cutaneous wound healing is a significant health issue in the US, often requiring skin grafts. StrataGraft (Stratatech Corporation, Madison, WI), a second-generation living human skin substitute created from NIKS human keratinocyte progenitors, was recently found to be a promising skin graft in phase I/II safety and efficacy clinical trial. NIKS proliferation is optimal in the presence of epidermal growth factor (EGF). Our preliminary data suggested that Notch signaling also plays a role in NIKS keratinocyte proliferation. Therefore, we hypothesized that EGF might stimulate NIKS proliferation by regulating Notch1 signaling. METHOD Notch1 messenger RNA (mRNA) levels from NIKS cells in monolayer culture were assessed by real-time polymerase chain reaction and Notch1 protein levels were detected by Western blot. To determine the role of EGF on Notch1 regulation, cells were incubated in basal media and then treated with EGF (10 ng/mL). A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to test NIKS cell proliferation. Cells were grown in basal media supplemented with EGF for 72 h in the presence or absence of N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) (0-30 μM), an inhibitor of Notch1 signaling. RESULTS Notch1 mRNA levels were cell confluence-dependent, being more abundant in a subconfluent cell monolayer. We detected a 2-fold decrease in Notch1 mRNA expression and a reduction in active Notch1 protein level in response to EGF. EGF treatment stimulated NIKS cellular proliferation. However, co-treatment with DAPT inhibited NIKS proliferation to basal levels. Blocking Notch1 activation by DAPT alone inhibited NIKS cellular proliferation (P < 0.01%). CONCLUSION Our results suggest that Notch1 is an essential downstream mediator of NIKS cellular proliferation via the EGF signaling pathway.