Rossitza Lazova

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.

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.

Autophagy in cutaneous malignant melanoma.

We show that malignant melanoma cells display high levels of autophagy, a cytoplasmic process of protein and organelle digestion that provides an energy source in times of nutrient deprivation. In a panel of 12 cases of cutaneous malignant melanoma of the superficial spreading type, cells in florid melanoma in situ (MIS) and invasive cells in the dermis appeared to be undergoing autophagy. Autophagosomes were detected through immunohistochemistry using the marker LC3B (microtubule‐associated light chain 3B), and by electron microscopy. Some autophagosomes contained melanized melanosomes, accounting for the phenomenon of ‘coarse melanin’ in malignant melanoma. Autophagosomes also contained the Golgi 58k protein, a structural component of the Golgi apparatus, and β1, 6‐branched oligosaccharides, indicating that at least some of the autophagosomal proteins were glycosylated with these structures. The findings suggest that autophagy could be a constitutive metabolic state for invasive and metastatic melanoma cells. Interestingly, a similar phenotype was also expressed by tumor‐associated melanophages. The findings are consistent with previous reports that endoplasmic reticulum (ER) stress drives melanoma progression, since ER stress is known to trigger autophagy. The results suggest that therapies inhibiting autophagy may be effective for the treatment of malignant melanoma by depriving cells of an important energy source.

Imaging mass spectrometry--a new and promising method to differentiate Spitz nevi from Spitzoid malignant melanomas.

Background: Differentiating Spitz nevus (SN) from Spitzoid malignant melanoma (SMM) is one the most difficult problems in dermatopathology. Specific Aim: To identify differences on proteomic level between SN and SMM. Methods: We performed Imaging Mass Spectrometry analysis on formalin-fixed, paraffin-embedded tissue samples to identify differences on proteomic level between SN and SMM. The diagnosis of SN and SMM was based on histopathologic criteria, clinical features, and follow-up data, which confirmed that none of the lesions diagnosed as SN recurred or metastasized. The melanocytic component (tumor) and tumor microenvironment (dermis) from 114 cases of SN and SMM from the Yale Spitzoid Neoplasm Repository were analyzed. After obtaining mass spectra from each sample, classification models were built using a training set of biopsies from 26 SN and 25 SMM separately for tumor and for dermis. The classification algorithms developed on the training data set were validated on another set of 30 samples from SN and 33 from SMM. Results: We found proteomic differences between the melanocytic components of SN and SMM and identified 5 peptides that were differentially expressed in the 2 groups. From these data, 29 of 30 SN and 26 of 29 SMM were recognized correctly based on tumor analysis in the validation set. This method correctly classified SN with 97% sensitivity and 90% specificity in the validation cohort. Conclusions: Imaging Mass Spectrometry analysis can reliably differentiate SN from SMM in formalin-fixed, paraffin-embedded tissue based on proteomic differences.

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.

Donor Y chromosome in renal carcinoma cells of a female BMT recipient: visualization of putative BMT-tumor hybrids by FISH.

Bone marrow-derived cells contribute to a wide variety of normal tissues such as liver, brain, and heart. This is due, at least in part, to cell fusion, in some cases with macrophages.1, 2, 3, 4, 5, 6 There are more than 25 reports of tumor hybridization in animals,7 and myeloid cell–tumor hybrids have been observed in melanoma,8 lymphoma,9 sarcoma,10, 11 and insulinoma models.12 In some cases, tumor hybridization was associated with metastasis.7 However, little is known of this in humans. Recently, the first genetic evidence for bone marrow–tumor cell hybridization in humans was reported.13 In that study, tumor DNA was analyzed from a child who developed metastatic renal cell carcinoma during a time when he had also received a BMT from his cancer-free, 6-year-old brother. Microdissected tumor cells of a nodal metastasis were shown through PCR to contain donor DNA, consistent with BMT–tumor hybridization.

Self Double-Stranded (ds)DNA Induces IL-1β Production from Human Monocytes by Activating NLRP3 Inflammasome in the Presence of Anti–dsDNA Antibodies

The pathogenic hallmark of systemic lupus erythematosus is the autoimmune response against self nuclear Ags, including dsDNA. The increased expression of the proinflammatory cytokine IL-1β has been found in the cutaneous lesion and PBMCs from lupus patients, suggesting a potential involvement of this cytokine in the pathogenesis of lupus. IL-1β is produced primarily by innate immune cells such as monocytes and can promote a Th17 cell response, which is increased in lupus. IL-1β production requires cleaving pro–IL-β into IL-1β by the caspase-1–associated multiprotein complex called inflammasomes. In this study we show that self dsDNA induces IL-1β production from human monocytes dependent on serum or purified IgG containing anti–dsDNA Abs by activating the nucleotide-binding oligomerization domain–like receptor family pyrin domain–containing 3 (NLRP3) inflammasome. Reactive oxygen species (ROS) and K+ efflux were involved in this activation. Knocking down the NLRP3 or inhibiting caspase-1, ROS, and K+ efflux decreased IL-1β production. Supernatants from monocytes treated with a combination of self dsDNA and anti–dsDNA Ab+ serum promoted IL-17 production from CD4+ T cells in an IL-1β–dependent manner. These findings provide new insights in lupus pathogenesis by demonstrating that self dsDNA together with its autoantibodies induces IL-1β production from human monocytes by activating the NLRP3 inflammasome through inducing ROS synthesis and K+ efflux, leading to the increased Th17 cell response.

Scar-localized argyria secondary to silver sulfadiazine cream.

Silver sulfadiazine cream is a topical antibacterial agent that combines the antibacterial effects of both silver and sulfadiazine. Its reported cutaneous side effects include hypersensitivity reactions, allergic contact dermatitis, erythema multiforme, and systemic argyria. We report the case of a patient who had localized argyria develop in a scar after the use of silver sulfadiazine cream. In this case, the silver sulfadiazine cream was applied to and argyria developed within a postsurgical wound and area of severe contact dermatitis.

Pseudoepitheliomatous hyperplasia: a review.

Pseudoepitheliomatous hyperplasia (PEH) is a benign condition, characterized by hyperplasia of the epidermis and adnexal epithelium, closely simulating squamous cell carcinoma. PEH may be present in a number of conditions characterized by prolonged inflammation and/or chronic infection, as well as in association with many cutaneous neoplasms. Herein, we review different inflammatory, infectious, and neoplastic skin diseases, in which florid epidermal hyperplasia is a prominent histopathologic feature, and introduce a systematic approach in the interpretation of PEH.

Cellular digital fibromas: distinctive CD34-positive lesions that may mimic dermatofibrosarcoma protuberans.

Background:  Digital fibromas are common benign acral tumors typically reported as angiofibromas (AFs) or acquired digital fibrokeratomas (ADFs). Cellular variants are not well recognized.