John M. Pawelek

PRODUCTION AND RELEASE OF PROOPIOMELANOCORTIN (POMC) DERIVED PEPTIDES BY HUMAN MELANOCYTES AND KERATINOCYTES IN CULTURE : REGULATION BY ULTRAVIOLET B

It is demonstrated that ultraviolet B (UVB) radiation stimulates increased expression of the proopiomelanocortin (POMC) gene which is accompanied by production and release of alpha-melanocyte stimulating hormone (alpha-MSH) and adrenocorticotropin (ACTH) by both normal and malignant human melanocytes and keratinocytes. The production and release of both peptides are also stimulated by dibutyryl cyclic adenosine monophosphate (dbcAMP) and interleukin 1 alpha (IL-1 alpha) but not by endothelin-1 (ET-1) or tumor necrosis factor-alpha (TNF-alpha). N-acetyl-cysteine (NAC), a precursor of glutathione (GSH), an intracellular free radical scavenger, abolishes the UVB-stimulated POMC peptide production and secretion. Conclusions are as follows: (1) Cultured human cells of cutaneous origin, namely keratinocytes and melanocytes, can produce and express POMC; (2) POMC expression is enhanced by exposure to UVB, possibly through a cyclic AMP-dependent pathway; and (3) The action of UVB on POMC production may involve a cellular response to oxidative stress.

Fusion of tumour cells with bone marrow-derived cells: a unifying explanation for metastasis.

The causes of metastasis remain elusive despite vast information on cancer cells. We posit that cancer cell fusion with macrophages or other migratory bone marrow-derived cells (BMDCs) provides an explanation. BMDC–tumour hybrids have been detected in numerous animal models and recently in human cancer. Molecular studies indicate that gene expression in such hybrids reflects a metastatic phenotype. Should BMDC–tumour fusion be found to underlie invasion and metastasis in human cancer, new approaches for therapy would surely follow.

Bacteria as tumour-targeting vectors

Live bacteria were first actively used in the treatment of cancer nearly 150 years ago, work that ultimately led to the study of immunomodulation. Today, with the discovery of bacterial strains that specifically target tumours, and aided by genomic sequencing and genetic engineering, there is new interest in the use of bacteria as tumour vectors. Bifodobacterium, Clostridium, and Salmonella have all been shown to preferentially replicate within solid tumours when injected from a distal site, and all three types of bacteria have been used to transport and amplify genes encoding factors such as prodrug-converting enzymes, toxins, angiogenesis inhibitors, and cytokines. In this review we provide a historical discussion of this area, and describe the development of the bacteria, which are currently being prepared for use in clinical trials in patients with cancer.

Targeting ER stress–induced autophagy overcomes BRAF inhibitor resistance in melanoma

Melanomas that result from mutations in the gene encoding BRAF often become resistant to BRAF inhibition (BRAFi), with multiple mechanisms contributing to resistance. While therapy-induced autophagy promotes resistance to a number of therapies, especially those that target PI3K/mTOR signaling, its role as an adaptive resistance mechanism to BRAFi is not well characterized. Using tumor biopsies from BRAF(V600E) melanoma patients treated either with BRAFi or with combined BRAF and MEK inhibition, we found that BRAFi-resistant tumors had increased levels of autophagy compared with baseline. Patients with higher levels of therapy-induced autophagy had drastically lower response rates to BRAFi and a shorter duration of progression-free survival. In BRAF(V600E) melanoma cell lines, BRAFi or BRAF/MEK inhibition induced cytoprotective autophagy, and autophagy inhibition enhanced BRAFi-induced cell death. Shortly after BRAF inhibitor treatment in melanoma cell lines, mutant BRAF bound the ER stress gatekeeper GRP78, which rapidly expanded the ER. Disassociation of GRP78 from the PKR-like ER-kinase (PERK) promoted a PERK-dependent ER stress response that subsequently activated cytoprotective autophagy. Combined BRAF and autophagy inhibition promoted tumor regression in BRAFi-resistant xenografts. These data identify a molecular pathway for drug resistance connecting BRAFi, the ER stress response, and autophagy and provide a rationale for combination approaches targeting this resistance pathway.

Biology of hypopigmentation

A review of the basics of pigment cell biology is followed by a discussion of the characteristics of several disorders of hypopigmentation. By determining such features as inheritance pattern, time of onset (congenital, childhood, adulthood), natural history (stable vs progressive), type of pigment loss (diffuse or circumscribed), distribution of lesions (generalized vs localized), degree of pigment loss (incomplete or complete), number of melanocytes, if any, in biopsy specimens of affected areas, type of melanocytic dysfunction, and associated inflammation or infection, one can classify the disorders of hypopigmentation. The proposed pathophysiology for each disorder of hypomelanosis is presented.

Punctate LC3B Expression Is a Common Feature of Solid Tumors and Associated with Proliferation, Metastasis, and Poor Outcome

Purpose: Measurement of autophagy in cancer and correlation with histopathologic grading or clinical outcomes has been limited. Accordingly, we investigated LC3B as an autophagosome marker by analyzing nearly 1,400 tumors from 20 types of cancer, focusing on correlations with clinical outcomes in melanoma and breast cancer. Experimental Design: Staining protocols were developed for automated quantitative analysis (AQUA) using antibodies versus LC3 isoform B (LC3B) and Ki-67. Clinically annotated breast and melanoma tissue microarrays (TMA) and a multitumor array were used. An AQUA program was developed to quantitate LC3B distribution in punctate and diffuse compartments of the cell. Results: LC3B staining was moderate to high in the large majority of tumors. The percentage of area occupied by punctate LC3B was elevated by 3- to 5-fold at high LC3B intensities. In breast cancer and melanoma TMAs, LC3B and Ki-67 showed strong correlations (P < 0.0001), and in multitumor TMAs, mitotic figures were most often seen in tumors with the highest LC3B expression (P < 0.002). In breast cancer, LC3B expression was elevated in node-positive versus node-negative primaries and associated with increased nuclear grade and shortened survival. In a melanoma TMA with no survival data, LC3B levels were highest in nodal, visceral, and cutaneous metastases. Conclusions: The results reveal a common expression of LC3B in malignancy and support emerging evidence that autophagy plays a significant role in cancer progression. High LC3B was associated proliferation, invasion and metastasis, high nuclear grade, and worse outcome. Thus, autophagy presents a key target of therapeutic vulnerability in solid tumors. Clin Cancer Res; 18(2); 370–9. ©2011 AACR.

The cancer cell--leukocyte fusion theory of metastasis.

The cause of metastasis remains elusive despite vast information on cancer cells. We posit that cancer cell fusion with macrophages or other migratory bone marrow-derived cells (BMDCs) provides an explanation. BMDCs fused with tumor cells were present in animal tumor xenografts where they were associated with metastases. In myeloma patients, transcriptionally active myeloma nuclei were incorporated into osteoclasts through fusion. In patients with renal cell carcinoma arising poststem cell transplant, donor genes were incorporated in recipient cancer cell nuclei, most likely through fusion, and showed tumor distribution patterns characteristic of cancer stem cells. Melanoma-macrophage hybrids generated in vitro contained chromosomes from both parental partners, showed increased ploidy, and transcribed and translated genes from both parents. They exhibited chemotactic migration in vitro toward fibronectin and exhibited high frequencies of metastasis when implanted in mice. They produced macromolecules that are characteristic of macrophages and known indicators of metastasis (c-Met, SPARC, MCR1, GnT-V, and the integrin subunits alpha(3), alpha(5), alpha(6), alpha(v), beta(1), beta(3)). They also produced high levels of beta1,6-branched oligosaccharides-predictors of poor survival in patients with melanoma or carcinomas of the breast, lung, and colon. We thus hypothesize that such gene expression patterns in cancer are generated through fusion. Tumor hybrids also showed active autophagy, a characteristic of both metastatic cancers and macrophages. BMDC-tumor cell fusion explains epidermal-mesenchymal transition in cancer since BMDCs express mesodermal traits and epithelial-mesenchymal transition regulators (Twist, SPARC, and others). If BMDC-tumor cell fusion underlies invasion and metastasis in human cancer, new approaches for therapeutic intervention would be mandated.

UVB-induced melanogenesis may be mediated through the MSH-receptor system.

Ultraviolet B radiation (UVB) elicits an increase in melanin production in mammalian skin. The mechanisms regulating this process are not understood, although it is well documented that there is an increase in the number of melanin-producing melanocytes. The melanotropins (MSH) are a family of peptides that increase the melanin content of melanocytes through an interaction with high affinity receptors. We have obtained evidence that the effects of UVB on melanogenesis may be mediated through an increase in MSH receptor activity on melanocytes. First, exposure of Cloudman S91 mouse melanoma cells to UVB resulted in increased binding of 125I-MSH to cells within 24 h. In five separate experiments, UVB-irradiated cultures displayed 2-10-fold increases in MSH binding capacity over that of unirradiated control cultures (optimum doses 10-20 mJ/cm2). Second, UVB and MSH potentiated one another in promoting cutaneous melanogenesis in both mice and guinea pigs. In the areas of guinea pig skin that received both UVB and MSH, there was a fivefold increase in active melanocytes/mm2 over the sum of active melanocytes/mm2 in areas receiving either MSH or UVB separately. Our results suggest that UVB light causes an increase in MSH receptor activity on cutaneous melanocytes, thus increasing cellular responsiveness to MSH. Implicit in this mechanism is a transduction of radiant energy into chemical energy during the process of UVB-induced melanogenesis.

Melanoma × macrophage hybrids with enhanced metastatic potential

Studies were conducted on the hypothesis that melanoma metastasis might be initiated through the gener-ation of hybrids comprised of cells of the primary tumor and tumor-infiltrating leukocytes. Fusion hybrids were generated in vitro between weakly metastatic Cloudman S91 mouse melanoma cells and normal mouse or human macrophages. Hybrids were implanted s.c. in the tail and mice were monitored for metastases. Controls included parental S91 cells, autologous S91 × S91 hybrids, and B16F10 melanoma cells. Of 35 hybrids tested, most were more aggressive than the parental melanoma cells, producing metastases sooner and in more mice. A striking characteristic was heterogeneity amongst hybrids, with some lines producing no metastases and others producing metastases in up to 80% of mice. With few exceptions, hybrids with the highest metastatic potential also had the highest basal melanin content whereas those with the lowest metastatic potential were basally amelanotic, as were t he parental melanoma cells. A spontaneous in vivo supermelanotic hybrid between an S91 tumor cell and DBA/2J host cell was one of the most metastatic lines. Hybrids with the highest metastatic potential also exhibited markedly higher chemotaxis to fibroblast-conditioned media. Histologically, the metastatic hybrids demonstrated vascular invasion and spread to distant organs similar to that of metastatic melanomas in mice and humans. Thus previous findings of enhanced metastasis in leukocyte × lymphoma hybrids can now be extended to include leukocyte × melanoma hybrids. Whether such hybridization is a natural cause of metastasis in vivo remains to be determined; however the fusion hybrids with genetically-matched parents described herein so closely resembled naturally- occurring metastatic melanoma cells that they could serve as useful new models for studies of this complex and deadly phenomenon.

Tumour-cell fusion as a source of myeloid traits in cancer

Malignant cells express molecular pathways that are also expressed by myeloid cells. Such behaviour is associated with loss of homotypic adhesion between cells, changes in the cellular matrix, induction of angiogenesis, motility, chemotaxis, and several immune-signalling pathways. The overlap between malignant cells and myeloid cells could be explained by one mechanism: fusion of myeloid cells and tumour cells, as noted in animal studies and in two patients with renal-cell carcinoma who underwent bone-marrow transplantation. An overlapping trait in these cells is their glycosylation patterns: hybrids have high expression of N-terminal glycosylation and beta1,6-branched oligosaccharides. In macrophages and cancer cells, these structures have a role in motility and systemic migration; in cancer, they are associated with metastasis and poor prognosis. In addition to myeloid traits, fusion might contribute to aneuploidy and plasticity in cancer. Understanding metastatic cells as myeloid-tumour hybrids suggests new strategies for diagnosis, treatment, and prevention of malignant disease.