Vincent Klump

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.

A Melanoma Brain Metastasis with a Donor-Patient Hybrid Genome following Bone Marrow Transplantation: First Evidence for Fusion in Human Cancer

Background Tumor cell fusion with motile bone marrow-derived cells (BMDCs) has long been posited as a mechanism for cancer metastasis. While there is much support for this from cell culture and animal studies, it has yet to be confirmed in human cancer, as tumor and marrow-derived cells from the same patient cannot be easily distinguished genetically. Methods We carried out genotyping of a metastatic melanoma to the brain that arose following allogeneic bone-marrow transplantation (BMT), using forensic short tandem repeat (STR) length-polymorphisms to distinguish donor and patient genomes. Tumor cells were isolated free of leucocytes by laser microdissection, and tumor and pre-transplant blood lymphocyte DNAs were analyzed for donor and patient alleles at 14 autosomal STR loci and the sex chromosomes. Results All alleles in the donor and patient pre-BMT lymphocytes were found in tumor cells. The alleles showed disproportionate relative abundances in similar patterns throughout the tumor, indicating the tumor was initiated by a clonal fusion event. Conclusions Our results strongly support fusion between a BMDC and a tumor cell playing a role in the origin of this metastasis. Depending on the frequency of such events, the findings could have important implications for understanding the generation of metastases, including the origins of tumor initiating cells and the cancer epigenome.

Expression of p16 alone does not differentiate between Spitz nevi and Spitzoid melanoma

Spitz nevi and Spitzoid melanomas show overlapping histopathologic features, often making the diagnosis challenging. The p16 protein functions as a tumor suppressor and loss of its expression may be seen in some melanomas.

An Unexpected Spectrum of p53 Mutations from Squamous Cell Carcinomas in Psoriasis Patients Treated with PUVA

Photochemotherapy employing 8‐methoxypsoralen and long‐wavelength ultraviolet radiation (UVA, 320‐400 nm) is widely used in the treatment of psoriasis. The pho‐toactivation of psoralens in skin cells leads to formation of DNA photoadducts which may be responsible, at least in part, for the efficacy of these photochemotherapies. However, mutations arising from these adducts may also lead to the well‐characterized increased incidence of squamous cell carcinoma. Mutations in the p53 tumor suppressor gene have been detected in many human cancers. To determine whether p53 mutations occur in squamous cell carcinomas in PUVA patients, PCR was used to amplify the exons (5‐9) in which other studies have found a high frequency of point mutations. Gel electrophoresis was used to detect single‐strand conformational polymorphisms. Aberrantly migrating bands were excised, reamplined and sequenced. Thirty‐four specimens from 10 patients were examined. Specimens from one patient who had received no phototherapy as well as from normal controls were also analyzed. Five of the 10 patients showed at least one p53 mutation. In contrast to previously reported psoralen‐induced p53 mutations in mice, the expected psoralen type mutations at alternating AT sites were not detected. All but two of the altered sequences occurred at dipyrimidine sites which is typical of solar type mutations. Two C→T mutations and two dipyrimidine mutations (CC→TT) were found. Other mutations included: C→G, G→T, C→A and an 18 bp deletion. A review of therapeutic history of these patients showed that some had also received UVB phototherapy. Furthermore, because sunlight is thought to be beneficial for psoriasis, nontherapeutic, casual UVB exposure cannot be excluded. Our observations suggest that the SCC may have arisen from the solar mutations and that PUVA may enhance tumor progression or immune suppression

Detecting HPV in cutaneous lesions using anti-HPV antibody immunohistochemistry.

Abstract:Most condyloma are diagnosed clinically (without a biopsy) or histopathologically (if biopsied) without any ancillary testing. In some cases, additional confirmation of productive infection by human papillomavirus (HPV) or typing of HPV is desired, and in situ hybridization (ISH) is the most commonly used test. However, ISH is not readily available in most laboratories and only detects certain genital subtypes of HPV. The aim of this study was to evaluate the sensitivity and specificity of an anti-HPV antibody, in 25 lesions (both HPV induced and non-HPV induced) mostly from the genital region, with comparison to results with ISH and findings on hematoxylin and eosin staining. The sensitivity and specificity for the anti-HPV antibody used in this study are 90.9% and 85.7%, respectively, compared with ISH. Immunohistochemistry with this anti-HPV antibody, like ISH, was generally positive in cases showing koilocytes/koilocytotic atypia (86%). Immunohistochemical staining also detected productive infection with HPV in 23% (3 of 13) of cases without koilocytes/koilocytotic atypia. Thus, although staining is generally positive in cases with diagnostic findings of koilocytes/koilocytotic atypia in hematoxylin and eosin sections, immunohistochemistry can detect HPV in some cases without koilocytes/koilocytotic atypia.

Neuropilin-2 as a useful marker in the differentiation between Spitzoid malignant melanoma and Spitz nevus

BACKGROUND Spitzoid malignant melanoma (SMM) shares many histopathologic features with Spitz nevus (SN). The distinction between SMM and SN remains one of the most difficult diagnostic problems in dermatopathology. Neuropilin-2 (NRP2) is a cytoplasmic/cell surface protein that is a mediator of melanoma-endothelial cell interaction. OBJECTIVE The aim of this study was to evaluate NRP2 expression in SMM and SN and to determine whether it can reliably differentiate between the 2 groups. METHODS We studied the expression of NRP2 in 19 cases of SMM and 19 cases of SN from Yale Spitzoid Neoplasm Repository, New Haven, Conn. RESULTS All 19 cases of SMM (100%) expressed NRP2. Most SMM showed moderate- and high-intensity staining in the majority of the melanoma cells. Most of the SN (14/19, 74%) were negative for the marker. NRP2 labeled only 5 of 19 SN (26%) and all of them demonstrated mild staining intensity. NRP2(+) staining was statistically significant in differentiating SMM from SN (P < .05). LIMITATIONS Small study size is a limitation. CONCLUSIONS NRP2 expression in SMM and SN may be a useful adjunct marker, in addition to histopathologic evaluation, in the differentiation between these 2 entities.

Evidence for glycosylation as a regulator of the pigmentary system: key roles of sialyl(α2-6)gal/GalNAc-terminated glycans in melanin synthesis and transfer

The major regulators of melanogenesis are glycoproteins, however no role for glycosylation in the pathway has yet been described. We stained skin biopsies and melanocyte-keratinocyte co-cultures with a panel of 20 lectins as oligosaccharide markers. Notably, the Elderberry Bark Lectin (EBL/SNA) stained melanocytes in both systems. EBL binds the sequence Neu5Ac(α(2-6)Gal/GalNAc)- at the termini of some oligosaccharide antennae. We used inhibitors of synthesis and/or binding of this sequence to assess effects on pigmentation. METHODS. Cell culture, lectin histochemistry, siRNA transfection, and assays for dopa oxidase and melanin were carried out by standard techniques. RESULTS. 6′-sialyllactose, a short homolog of the sequence in question, anti-sialyltransferase 6 (ST6) siRNA, and cytidine, a sialyltransferase (ST) inhibitor, each inhibited EBL binding, melanogenesis and melanosome transfer. Unexpectedly, 3′-sialyllactose and siRNA for ST3, chosen as a negative controls, also inhibited these processes. Though strong inhibitors of melanization, none of the agents affected tyrosinase/dopa oxidase activity, indicating previously unrecognized post-tyrosinase regulation of melanization. CONCLUSIONS. We report for the first time that Neu5Ac (α(2-6)Gal/GalNAc)- and possibly Neu5Ac(α(2–3)Gal/GalNAc)-terminated oligosaccharides play multiple roles in melanin synthesis and transfer.

Abstract 3786: LC3B-positive autophagosomes are a common feature of solid tumors and associated with proliferation, metastasis and poor outcome

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Interest in autophagy and cancer has surged in recent years. Autophagy provides a source of nutrients in times of metabolic stress. A paradox for autophagy in cancer is that while autophagy is a conserved pro-survival mechanism, it can also act in tumor suppression as is the case with the autophagy-associated beclin-1 protein. However, there is growing evidence supporting a pro-survival role for autophagy in tumor progression, for example in gastrointestinal tumors and invasive melanomas wherein autophagosomes are abundant. Here we expanded previous studies on autophagosomes in malignancies to include nearly 2000 pathology specimens from 20 different cancers, analyzing for associations of autophagosomes with tumor progression. Methods. Individual pathology specimens and tumor tissue microarrays (TMAs) were stained for the autophagosome marker LC3B and the proliferation marker Ki-67 through standard IHC procedures. Automated quantitative analyses (AQUA) used fluorescence-tagged antibodies. Results: In multitumor TMAs, about 85% of all tumors showed positive staining of LC3B in a granular pattern. In melanoma TMAs, LC3B was elevated in lymph node metastases. In breast carcinoma TMAs LC3B was elevated in node-positive compared to node-negative primaries and associated with increased nuclear grade, and worse patient outcome. LC3B and Ki-67 expression correlated in both melanoma and breast carcinoma (p < 0.0001), indicating a strong association between autophagy and proliferation. Supporting this, mitotic figures were largely detected in LC3B-positive tumor cells. Conclusions. While it is clear that autophagy can play a tumor suppressor role in some settings, we posit that these results are most consistent with a pro-survival role for autophagy, at least in advanced malignancies. In cancer, autophagy is thought to be part of the pro-survival integrated stress response known as aerobic glycolysis or the Warburg effect whereby cells utilize glycolysis rather than aerobic respiration for ATP production. Since some 90% of malignancies constituitively express the Warburg effect and, as shown herein, a similar number express LC3B-positive autophagosomes, the results are most consistent with autophagy being an integral, active part of this response. That autophagosomes are expressed in proliferating tumor cells further supports this proposal. In any case, the widespread occurrence of autophagosomes in cancer shown herein, and by others, underscores the importance of autophagy as a target in the design of new anti-cancer therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3786. doi:10.1158/1538-7445.AM2011-3786