Targeted radionuclide therapy of melanoma: Anti‐tumoural efficacy studies of a new 131I labelled potential agent

In recent years, there has been dramatic worldwide increase in incidence of malignant melanoma. Although localised disease is often curable by surgical excision, metastatic melanoma is inherently resistant to most treatments. In this context, targeted radionuclide therapy could be an efficient alternative. After pharmacomodulation study, we selected a quinoxaline derivative molecule (ICF01012) for its high, specific and long‐lasting uptake in melanoma with rapid clearance from nontarget organs providing suitable dosimetry parameters for targeted radiotherapy. Aim of this study was to investigate, in vivo, efficacy of [¹³¹I]ICF01012 on nonmetastatic B16F0, metastatic B16Bl6 or human M4Beu melanoma tumours. First, colocalisation of ICF01012 with melanin by SIMS imaging was observed. Second, we showed that treatment drastically inhibited growth of B16F0, B16Bl6 and M4beu tumours whereas [¹³¹I]NaI or unlabelled ICF01012 treatment was without significant effect. Histological analysis and measure of PCNA proliferation marker expression showed that residual B16 tumour cellsexhibit a significant loss of aggressiveness after treatment. This effect is associated with a lengthening of the treated‐mice survival time. Moreover, with B16Bl6 model, 55% of the untreated mice had lung metastases whereas no metastasis was counted on treated group. Our data demonstrated a strong anti‐tumoural effect of [¹³¹I]ICF01012 for radionuclide therapy on murine and human in vivo pigmented melanoma models, whatever their dissemination profiles and their melanin content be. Further studies will attempt to optimise therapy protocol by increasing the balance between the anti‐tumoural effect and the safety on nontarget organs. © 2009 UICC     

Bcl-X(L) is a chemoresistance factor in human melanoma cells that can be inhibited by antisense therapy

Malignant melanoma is a tumor that responds poorly to a variety of apoptosis-inducing treatment modalities, such as chemotherapy. The expression of genes that regulate apoptotic cell death plays an important role in determining the sensitivity of tumor cells to chemotherapeutic intervention. Bcl-x(L) is an antiapoptotic member of the Bcl-2 family and is universally expressed in human melanoma. To evaluate the Bcl-x(L) protein as a potential therapeutic target in melanoma, the influence of Bcl-x(L) expression levels on the chemoresistance of human melanoma cells was investigated. Overexpression of Bcl-x(L) in stably transfected human melanoma Mel Juso cells significantly reduced sensitivity to cisplatin-induced apoptosis (p < or = 0.05). In a parallel approach, reduction of Bcl-x(L) protein by specific AS oligonucleotide (ISIS 16009) treatment enhanced the chemosensitivity of Mel Juso cells by 62% compared to cells treated with MM control oligonucleotide (ISIS 16967) as well as chemotherapy-induced apoptosis. These data suggest that Bcl-x(L) is an important factor contributing to the chemoresistance of human melanoma. Reduction of Bcl-x(L) expression by AS oligonucleotides provides a rational and promising approach that may help to overcome chemoresistance in this malignancy.     

Personal history of non‐melanoma skin cancer diagnosis and death from melanoma in women

Melanoma incidence is increasing. We evaluated risk of melanoma death after diagnosis of non‐melanoma skin cancer (NMSC). We followed 77,288 female American nurses from the Nurses’ Health Study from 1986 to 2012. We used Cox proportional hazards models to determine the hazard ratio (HR) of lethal and non‐lethal melanoma diagnosis and melanoma death, according to personal NMSC history. Among melanoma cases, we examined the HR of melanoma death and the odds ratio (OR) of melanoma with a Breslow thickness ≥0.8 mm or Clark's levels of IV and V according to history of NMSC. We documented 930 melanoma cases without NMSC history and 615 melanoma cases with NMSC history over 1.8 million person‐years. The multivariate‐adjusted HR (95% confidence interval) of melanoma death associated with personal history of NMSC was 2.89 (1.85–4.50). Women with history of NMSC were more likely to develop non‐lethal melanoma than lethal melanoma (HR (95% CI): 2.31 (2.05–2.60) vs. 1.74 (1.05–2.87)). Among melanoma cases, women with history of NMSC had a non‐significant decreased risk of melanoma deaths (0.87 (0.55–1.37)), Breslow thickness ≥0.8 mm (0.85 (0.59–1.21)) and Clark's levels IV and V (0.81(0.52–1.24)). Women with NMSC history were less likely to be diagnosed with a lethal melanoma than a non‐lethal melanoma, but overall rate of melanoma diagnosis was increased in both subtypes, leading to the increased risk of melanoma death. Our findings suggest the continued need for dermatologic screening for patients after NMSC diagnosis, given increased melanoma risk. Early detection among NMSC patients may decrease deaths from melanoma.     

Caveolin‐1 tumor‐promoting role in human melanoma

Caveolin‐1 (Cav‐1), a member of the caveolin family, regulates caveolae‐associated signaling proteins, which are involved in many biological processes, including cancer development. Cav‐1 was found to exert a complex and ambiguous role as oncogene or tumor suppressor depending on the cellular microenvironment. Here we investigated Cav‐1 expression and function in a panel of melanomas, finding its expression in all the cell lines. The exception was the primary vertical melanoma cell line, WM983A, characterized by the lack of Cav‐1, and then utilized as a recipient for Cav‐1 gene transduction to address a series of functional studies. The alleged yet controversial role of phospho (Ph)‐Cav‐1 on cell regulation was also tested by transducing the nonphosphorylatable Cav‐1Y14A mutant. Wild‐type Cav‐1, but not mutated Cav‐1Y14A, increased tumorigenicity as indicated by enhanced proliferation, migration, invasion and capacity of forming foci in semisolid medium. Accordingly, Cav‐1 silencing inhibited melanoma cell growth reducing some of the typical traits of malignancy. Finally, we detected a secreted fraction of Cav‐1 associated with cell released microvesicular particles able to stimulate in vitro anchorage independence, migration and invasion in a paracrine/autocrine fashion and, more important, competent to convey metastatic asset from the donor melanoma to the less aggressive recipient cell line. A direct correlation between Cav‐1 levels, the amount of microvesicles released in the culture medium and MMP‐9 expression was also observed. © 2009 UICC     

Phase II trial of sagopilone, a novel epothilone analog in metastatic melanoma

Background:

Sagopilone is a novel fully synthetic epothilone with promising preclinical activity and a favourable toxicity profile in phase I testing.

Methods:

A phase II pharmacokinetic and efficacy trial was conducted in patients with metastatic melanoma. Patients had measurable disease, Eastern Cooperative Oncology Group performance status 0–2, adequate haematological, and organ function, with up to 2 previous chemotherapy and any previous immunotherapy regimens. Sagopilone, 16 mg m⁻², was administered intravenously over 3 h every 21 days until progression or unacceptable toxicity.

Results:

Thirty-five patients were treated. Sagopilone showed multi-exponential kinetics with a mean terminal half-life of 64 h and a volume of distribution of 4361 l m⁻² indicating extensive tissue/tubulin binding. Only grade 2 or lower toxicity was observed: these included sensory neuropathy (66%), leukopenia (46%), fatigue (34%), and neutropenia (31%). The objective response rate was 11.4% (one confirmed complete response, two confirmed partial responses, and one unconfirmed partial response). Stable disease for at least 12 weeks was seen in an additional eight patients (clinical benefit rate 36.4%).

Conclusion:

Sagopilone was well tolerated with mild haematological toxicity and sensory neuropathy. Unlike other epothilones, it shows activity against melanoma even in pretreated patients. Further clinical testing is warranted.     

Leiodermatolide,a novel marine natural product,has potent cytotoxic and antimitotic activity against cancer cells,appears to affect microtubule dynamics,and exhibits antitumor activity

Pancreatic cancer, the fourth leading cause of cancer death in the United States, has a negative prognosis because metastasis occurs before symptoms manifest. Leiodermatolide, a polyketide macrolide with antimitotic activity isolated from a deep water sponge of the genus Leiodermatium, exhibits potent and selective cytotoxicity toward the pancreatic cancer cell lines AsPC‐1, PANC‐1, BxPC‐3, and MIA PaCa‐2, and potent cytotoxicity against skin, breast and colon cancer cell lines. Induction of apoptosis by leiodermatolide was confirmed in the AsPC‐1, BxPC‐3 and MIA PaCa‐2 cells. Leiodermatolide induces cell cycle arrest but has no effects on in vitro polymerization or depolymerization of tubulin alone, while it enhances polymerization of tubulin containing microtubule associated proteins (MAPs). Observations through confocal microscopy show that leiodermatolide, at low concentrations, causes minimal effects on polymerization or depolymerization of the microtubule network in interphase cells, but disruption of spindle formation in mitotic cells. At higher concentrations, depolymerization of the microtubule network is observed. Visualization of the growing microtubule in HeLa cells expressing GFP‐tagged plus end binding protein EB‐1 showed that leiodermatolide stopped the polymerization of tubulin. These results suggest that leiodermatolide may affect tubulin dynamics without directly interacting with tubulin and hint at a unique mechanism of action. In a mouse model of metastatic pancreatic cancer, leiodermatolide exhibited significant tumor reduction when compared to gemcitabine and controls. The antitumor activities of leiodermatolide, as well as the proven utility of antimitotic compounds against cancer, make leiodermatolide an interesting compound with potential chemotherapeutic effects that may merit further research.     

Alpha fetoprotein is a novel protein‐binding partner for caspase‐3 and blocks the apoptotic signaling pathway in human hepatoma cells

Although there is increasing evidence that alpha fetoprotein (AFP) may function as regulatory factor in the growth of tumor cells, the precise mechanism is still unclear. In the current study, we investigated the role of the cytoplasmic AFP in caspase‐3‐mediated signaling of apoptosis. Our results showed that low doses of TNF‐related apoptosis‐inducing ligand (TRAIL) elevated the activity of caspase‐8, but not caspase‐3. Caspase‐3 colocalized and interacted with AFP in the cytoplasm of Bel 7402 cells, and translocated into nuclei in association with the occurrence of apoptosis while cells were under cotreatment with all‐trans retinoic acid (ATRA) or TRAIL. AFP was able to form complexes with caspase‐3 and block onward transmission of signaling from caspase‐8. Knockdown of AFP increased the sensitivity of Bel 7402 cells to TRAIL, and thereby, triggered caspase‐3 signaling. No intermolecule interaction occurred between AFP and caspase‐8, nor was caspase‐8 activity altered after AFP knockdown, demonstrating the selectivity of AFP in interfering with the apoptotic signaling pathway. The effect of AFP on caspase‐3 was further confirmed by transfection of the AFP gene into HLE cells (AFP negative). We conclude that ATRA or TRAIL resistance in AFP producing hepatoma is at least, in part, attributable to the high level of the cytoplasmic AFP. Therefore, it is possible that the combination of AFP gene silencing together with ATRA/TRAIL cotreatment will benefit the enhancement of the chemotherapeutic efficiency of these agents on tumors. © 2009 UICC     

IGFBP‐3 inhibits Wnt signaling in metastatic melanoma cells

In previous works, we have shown that insulin‐like growth factor‐binding protein‐3 (IGFBP‐3), a tissue and circulating protein able to bind to IGFs, decreases drastically in the blood serum of patients with diffuse metastatic melanoma. In agreement with the clinical data, recombinant IGFBP‐3 was found to inhibit the motility and invasiveness of cultured metastatic melanoma cells and to prevent growth of grafted melanomas in mice. The present work was aimed at identifying the signal transduction pathways underlying the anti‐tumoral effects of IGFBP‐3. We show that the anti‐tumoral effect of IGFBP‐3 is due to inhibition of the Wnt pathway and depends upon the presence of CD44, a receptor protein known to modulate Wnt signaling. Once it has entered the cell, IGFBP‐3 binds the Wnt signalosome interacting specifically with its component GSK‐3β. As a consequence, the β‐catenin destruction complex dissociates from the LRP6 Wnt receptor and GSK‐3β is activated through dephosphorylation, becoming free to target cytoplasmic β‐catenin which is degraded by the proteasomal pathway. Altogether, the results suggest that IGFBP‐3 is a novel and effective inhibitor of Wnt signaling. As IGFBP‐3 is a physiological protein which has no detectable toxic effects either on cultured cells or live mice, it might qualify as an interesting new therapeutic agent in melanoma, and potentially many other cancers with a hyperactive Wnt signaling. © 2016 The Authors. Molecular Carcinogenesis Published by Wiley Periodicals, Inc.     

Bromodomain-containing protein 4 inhibitor JQ1 promotes melanoma cell apoptosis by regulating mitochondrial dynamics

Although the role of bromodomain-containing protein 4 (BRD4) in ovarian cancer, pancreatic cancer, lymphoma, and many other diseases is well known, its function in cutaneous melanoma is only partially understood. The results of the present study show that the BRD4 inhibitor JQ1 promotes the apoptosis of B16 melanoma cells by altering mitochondrial dynamics, thereby inducing mitochondrial dysfunction and increasing oxidative stress. We found that treatment of B16 cells with different concentrations of JQ1 (125 nmol/L or 250 nmol/L) significantly downregulated the expression of protein subunits involved in mitochondrial respiratory chain complexes I, III, IV, and V, increased reactive oxygen species, induced energy metabolism dysfunction, significantly enhanced apoptosis, and activated the mitochondrial apoptosis pathway. At the same time, JQ1 inhibited the activation of AMP-activated protein kinase, a metabolic energy sensor. In addition, we found that the mRNA and protein levels of mitochondrial dynamin-related protein 1 increased, whereas the levels of mitochondrial fusion protein 1 and optic atrophy protein 1 decreased. Mechanistically, we determined that JQ1 inhibited the expression of c-Myc and altered mitochondrial dynamics, eventually leading to changes in the mitochondrial function, metabolism, and apoptosis of B16 melanoma cells.     

Integrating tumor‐initiating cells into the paradigm for melanoma targeted therapy

There is growing evidence to suggest that not all cancer cells have similar levels of malignant potential and that tumor progression may be driven by specialized sub‐sets of “tumor initiating” cells. It is likely that as tumor initiating cells have lower proliferation rates and enhanced survival mechanisms they may also drive drug resistance. Melanoma is known to be an exceptionally therapy resistant tumor, with no treatment yet identified to alter the natural progression of the disseminated disease. In the current review, we discuss evidence for the existence of melanoma initiating cells and described possible therapeutic strategies to eradicate this population via the targeting of specific cell‐surface markers or through the disruption of the interaction of the melanoma initiating cells with their local microenvironment. It is hoped that the targeting of melanoma initiating cells may be one approach to overcome the incredible therapy resistance of this tumor. © 2008 Wiley‐Liss, Inc.     

Results of a phase II,open‐label,non‐comparative study of intralesional PV‐10 followed by radiotherapy for the treatment of in‐transit or metastatic melanoma

BACKGROUND

In‐transit and recurrent dermal or subcutaneous melanoma metastases represent a significant burden of advanced disease. Intralesional Rose Bengal can elicit tumor selective ablation and a T‐cell mediated abscopal effect in untreated lesions. A subset of patients in a phase II trial setting received external beam radiotherapy to their recurrent lesions with complete or partial response and no significant acute radiation reaction.

METHODS

An open‐label, single‐arm phase II study was performed to assess the efficacy and safety of PV‐10 followed by hypofractionated radiotherapy. Patients had in‐transit melanoma metastases suitable for IL therapy and radiotherapy.

RESULTS

Fifteen patients were enrolled and thirteen completed both treatment components. The overall response rate was 86.6% and the clinical benefit was 93.3% on an intention to treat analysis (CR 33.3%, PR 53.3%, SD 6.7%). The median follow up duration was 19.25 months. Size of metastases (<10 mm) predicted lesion complete response (74.6%). Treatment was well tolerated with no associated grade 4 or 5 adverse events.

CONCLUSIONS

The combination of PV‐10 and radiotherapy resulted in lesion‐specific, normal tissue‐sparing, ablation of disease with minimal local or systemic adverse effects.