Ashok K. Chakraborty

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.

Proopiomelanocortin, corticotropin releasing hormone and corticotropin releasing hormone receptor genes are expressed in human skin

Evidence is provided that human skin, the largest body organ exposed to multiple stressors, expresses proopiomelanocortin (POMC), corticotropin releasing hormone (CRH) and CRH‐receptor (CRHR) genes in vivo. In vitro studies show that POMC and CRHR mRNAs are transcribed in melanocytes, cells derived from the neural crest, and in keratinocytes, cells derived from the ectoderm. CRH mRNA is transcribed in cultured melanocytes but not in keratinocytes. It is proposed that an equivalent of the ‘hypothalamus‐pituitary axis’ composed of the CRH‐CRHR‐POMC loop is conserved in mammalian skin.

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.

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.

UV Light and MSH Receptors

ABSTRACT: Ultraviolet B (UVB) radiation in the skin induces pigmentation that protects cells from further UVB damage and reduces photocarcinogenesis. Although the mechanisms are not well understood, our laboratory has shown that UVB radiation causes increased MSH receptor activity by redistributing MSH receptors from internal pools to the external surface, with a resultant increase in cellular responsiveness to MSH. By this means, UVB and MSH act synergistically to increase melanin content in the skin of mice and guinea pigs. In humans, MSH causes increased skin pigmentation, predominantly in sun‐exposed areas. We have shown recently that UVB irradiation and exposure to MSH or to dbcAMP, stimulates production of mRNAs for both αMSH receptors and POMC in human melanocytes and keratinocytes. This indicates that at least one action of UVB on the pigmentary system is mediated through increased MSH receptor production, as well as through the production of the signal peptides, MSH and ACTH, that can further activate MSH receptors. The results add support to the hypothesis that the effects of UVB on cutaneous melanogenesis are mediated through a series of coordinated events in which MSH receptors and POMC‐derived peptides play a central role.

Genotype-Selective Combination Therapies for Melanoma Identified by High-Throughput Drug Screening

UNLABELLED Resistance and partial responses to targeted monotherapy are major obstacles in cancer treatment. Systematic approaches to identify efficacious drug combinations for cancer are not well established, especially in the context of genotype. To address this, we have tested pairwise combinations of an array of small-molecule inhibitors on early-passage melanoma cultures using combinatorial drug screening. Results reveal several inhibitor combinations effective for melanomas with activating RAS or BRAF mutations, including mutant BRAF melanomas with intrinsic or acquired resistance to vemurafenib. Inhibition of both EGF receptor and AKT sensitized treatment-resistant BRAF mutant melanoma cultures to vemurafenib. Melanomas with RAS mutations were more resistant to combination therapies relative to BRAF mutants, but were sensitive to combinations of statins and cyclin-dependent kinase inhibitors in vitro and in vivo. These results show the use of combinatorial drug screening for discovering unique treatment regimens that overcome resistance phenotypes of mutant BRAF- and RAS-driven melanomas. SIGNIFICANCE We have used drug combinatorial screening to identify effective combinations for mutant BRAF melanomas, including those resistant to vemurafenib, and mutant RAS melanomas that are resistant to many therapies. Mechanisms governing the interactions of the drug combinations are proposed, and in vivo xenografts show the enhanced benefit and tolerability of a mutant RAS -selective combination, which is currently lacking in the clinic.

Ultraviolet B stimulates production of corticotropin releasing factor (CRF) by human melanocytes

Here we show that human melanocytes express the corticotropin releasing factor (CRF) gene and produce CRF peptide. The CRF production and secretion is markedly stimulated by ultraviolet B (UVB) radiation. This is the first demonstration that cutaneous melanocytes respond to environmental stress (UVB) through the production of CRF.

Donor DNA in a renal cell carcinoma metastasis from a bone marrow transplant recipient.

Individuals receiving either allogeneic bone marrow transplants, or organ transplants, are at an increased risk of developing de novo malignancies. Radiation and immunosuppression are both risk factors. As unfortunate as this is, it could allow for detection of tumor cell–hematopoietic cell hybrids in human cancer. In animal models, tumor hybrids were documented through the use of heterologous genetic markers for the tumor and host genotypes, in some cases revealing hybridization with hematopoietic cells. In noncancer systems, heterologous markers were used to demonstrate fusion of bone marrow-derived stem cells with tissue cells. We looked for BMT donor DNA in a paraffin-embedded metastasis from a child who, after allogeneic liver and bone marrow transplants, developed renal cell carcinoma, and then metastases. The pathology report described this specimen as showing portions of a right caval lymph node involved with metastatic renal cell carcinoma. The primary tumor was unavailable. Both the patient and his liver donor were immunotyped as Oþ ; the BMT donor was Aþ . Laser microdissection, sample preparation, and PCR were as described. Two primer pairs were designed to distinguish between A and O alleles (Table 1A). The primer pairs were redundant and were used in different reaction tubes. Restriction fragments were generated by KpnI digestion. In all, 14 of the 21 tumor samples were microdissected by a pathologist (RL), and seven under the supervision of a pathologist (FP). For a BMT recipient, any blood lineage cells in the tumor will be of donor genotype; thus, it is important to note that the tumor contained fields in which tumor cells could readily be microdissected free of normal cells. Carcinoma cells were distinguished by their large nuclei as compared to normal cells with smaller nuclei (Figure 1). Buccal DNA samples from donor and recipient were subjected to PCR with both 176 and 193bp primers (Table 1A). Following KpnI, amplified donor DNA yielded bands of 176bp (A allele) and 150bp (O allele) from the 176bp primers (Figure 2a, upper); and 193bp (A allele) and 174bp (O allele) from the 193bp primers (Figure 2a, lower). In contrast, KpnI digestion of recipient DNA yielded only 150 and 174bp bands from the respective primer pairs (O allele). Thus, the donor genotype was A/O, and the recipient was O/O, consistent with the clinical immunotyping. Tumor cell DNA was amplified using the 176bp primers, aliquots were subjected to KpnI digestion, and the mixtures were run on agarose gels. Results for tumor samples T2 and T6 are in Figure 2b. Without KpnI ( ), a single 176bp band was seen, whereas with KpnI (þ ) two bands of 176 and 150bp were seen, consistent with being A and O allele fragments. Similarly, amplification of tumor samples T5 and T6 with the 193bp primers and KpnI digestion yielded bands of 193 and 174bp, as predicted for A and O alleles (Figure 2c). KpnI digestion of tumor sample T19 produced a 193bp A allele fragment, but no O allele fragment (Figure 2c). In a second experiment, tumor samples T7–T14 were amplified with the 176 bp primers and run on an agarose gel without KpnI. The 176 bp fragments were isolated by needle prick, re-amplified with the 176 bp primers, digested with KpnI, and run again on a gel (Figure 2d). After KpnI, all tumor samples showed both 176 bp (A allele) and 150 bp (O allele) fragments. This was confirmed by sequencing of KpnI-generated 176 and 150 bp DNA fragments from tumor DNA sample T8, from the gel in Figure 2d (Table 1B). The sequences of the 176 and 150 bp bands from tumor sample T8 were identical to the published sequences for the A and O alleles, as were the corresponding bands of donor DNA. To show that the putative A allele bands were truly KpnIresistant, and not due to incomplete KpnI digestion, previously digested 176 bp fragments from donor and tumor DNA were isolated from gels via needle prick and re-amplified. Aliquots were either mock-digested or digested a second time with KpnI. The once-digested (þ ) and twice-digested (þ þ ) samples were again run on a gel (Figure 2e). After the second KpnI digestion, donor DNA and tumor DNA samples T2, T3, T4, and T5 showed only the 176 bp product, and no further 150 bp was generated. This indicated that the original KpnI digestion had been complete, and that the remaining 176 bp fragments were truly KpnI resistant, consistent with the tumor cells containing the A allele.

Co-Targeting Insulin-Like Growth Factor I Receptor and HER2: Dramatic Effects of HER2 Inhibitors on Nonoverexpressing Breast Cancer

The insulin-like growth factor I receptor (IGFIR) and HER2 display important signaling interactions in breast cancer. We examined the effect of combinations of antagonists of these receptors using two human breast cancer cell lines: BT474 (HER2+, IGFIR low) and MCF7 (HER2 low, IGFIR high). In BT474 cells, growth was inhibited by HER2 antagonists but not by IGFIR antagonists; however, IGFIR antagonists enhanced the effect of HER2 inhibitors. In MCF7 cells, growth was inhibited by IGFIR antagonists but not by HER2 antagonists; however, HER2 antagonism enhanced the effect of IGFIR inhibitors. Synergistic inhibition of soft agar growth was also observed. Although HER2 and IGFIR antagonists individually only minimally affected cell cycle, their combination gave a small enhancement of their effects. No single receptor-targeting drug was capable of inducing apoptosis, but combining antagonists of both receptors induced a dramatic degree of apoptosis in both cell lines. Induction of apoptosis was most striking in MCF7 cells using a Herceptin/IGFIR antagonist combination despite these cells being HER2 nonoverexpressing. Toward understanding the mechanism of these effects, we detected coassociation IGFIR and HER2 in both cell lines. Specific inhibitors of one of these receptors could cross-inhibit the activity of the other. Targeting both receptors gave the maximal inhibition of their downstream extracellular signal-regulated kinase 1/2 and AKT signaling pathways. Hence, such drug combinations may be clinically useful and may be beneficial even in tumors in which single drugs are inactive, as exemplified by the effect of the HER2/IGFIR inhibitor combination in HER2 nonoverexpressing MCF7 cells.