Anatomic position determines oncogenic specificity in melanoma.

Oncogenic alterations to DNA are not transforming in all cellular contexts1,2. This may be due to pre-existing transcriptional programmes in the cell of origin. Here we define anatomic position as a major determinant of why cells respond to specific oncogenes. Cutaneous melanoma arises throughout the body, whereas the acral subtype arises on the palms of the hands, soles of the feet or under the nails3. We sequenced the DNA of cutaneous and acral melanomas from a large cohort of human patients and found a specific enrichment for BRAF mutations in cutaneous melanoma and enrichment for CRKL amplifications in acral melanoma. We modelled these changes in transgenic zebrafish models and found that CRKL-driven tumours formed predominantly in the fins of the fish. The fins are the evolutionary precursors to tetrapod limbs, indicating that melanocytes in these acral locations may be uniquely susceptible to CRKL. RNA profiling of these fin and limb melanocytes, when compared with body melanocytes, revealed a positional identity gene programme typified by posterior HOX13 genes. This positional gene programme synergized with CRKL to amplify insulin-like growth factor (IGF) signalling and drive tumours at acral sites. Abrogation of this CRKL-driven programme eliminated the anatomic specificity of acral melanoma. These data suggest that the anatomic position of the cell of origin endows it with a unique transcriptional state that makes it susceptible to only certain oncogenic insults.

Cooperation between melanoma cell states promotes metastasis through heterotypic cluster formation.

Melanomas can have multiple coexisting cell states, including proliferative (PRO) versus invasive (INV) subpopulations that represent a "go or grow" trade-off; however, how these populations interact is poorly understood. Using a combination of zebrafish modeling and analysis of patient samples, we show that INV and PRO cells form spatially structured heterotypic clusters and cooperate in the seeding of metastasis, maintaining cell state heterogeneity. INV cells adhere tightly to each other and form clusters with a rim of PRO cells. Intravital imaging demonstrated cooperation in which INV cells facilitate dissemination of less metastatic PRO cells. We identified the TFAP2 neural crest transcription factor as a master regulator of clustering and PRO/INV states. Isolation of clusters from patients with metastatic melanoma revealed a subset with heterotypic PRO-INV clusters. Our data suggest a framework for the co-existence of these two divergent cell populations, in which heterotypic clusters promote metastasis via cell-cell cooperation.

Developmental chromatin programs determine oncogenic competence in melanoma.

Oncogenes only transform cells under certain cellular contexts, a phenomenon called oncogenic competence. Using a combination of a human pluripotent stem cell­derived cancer model along with zebrafish transgenesis, we demonstrate that the transforming ability of BRAFV600E along with additional mutations depends on the intrinsic transcriptional program present in the cell of origin. In both systems, melanocytes are less responsive to mutations, whereas both neural crest and melanoblast populations are readily transformed. Profiling reveals that progenitors have higher expression of chromatin-modifying enzymes such as ATAD2, a melanoma competence factor that forms a complex with SOX10 and allows for expression of downstream oncogenic and neural crest programs. These data suggest that oncogenic competence is mediated by regulation of developmental chromatin factors, which then allow for proper response to those oncogenes.

The Stress-Like Cancer Cell State Is a Consistent Component of Tumorigenesis.

Transcriptional profiling of tumors has revealed a stress-like state among the cancer cells with the concerted expression of genes such as fos, jun, and heat-shock proteins, though this has been controversial given possible dissociation-effects associated with single-cell RNA sequencing. Here, we validate the existence of this state using a combination of zebrafish melanoma modeling, spatial transcriptomics, and human samples. We found that the stress-like subpopulation of cancer cells is present from the early stages of tumorigenesis. Comparing with previously reported single-cell RNA sequencing datasets from diverse cancer types, including triple-negative breast cancer, oligodendroglioma, and pancreatic adenocarcinoma, indicated the conservation of this state during tumorigenesis. We also provide evidence that this state has higher tumor-seeding capabilities and that its induction leads to increased growth under both MEK and BRAF inhibitors. Collectively, our study supports the stress-like cells as a cancer cell state expressing a coherent set of genes and exhibiting drug-resistance properties.

Regulation of the error-prone DNA polymerase Polκ by oncogenic signaling and its contribution to drug resistance.

The DNA polymerase Polκ plays a key role in translesion synthesis, an error-prone replication mechanism. Polκ is overexpressed in various tumor types. Here, we found that melanoma and lung and breast cancer cells experiencing stress from oncogene inhibition up-regulated the expression of Polκ and shifted its localization from the cytoplasm to the nucleus. This effect was phenocopied by inhibition of the kinase mTOR, by induction of ER stress, or by glucose deprivation. In unstressed cells, Polκ is continually transported out of the nucleus by exportin-1. Inhibiting exportin-1 or overexpressing Polκ increased the abundance of nuclear-localized Polκ, particularly in response to the BRAFV600E-targeted inhibitor vemurafenib, which decreased the cytotoxicity of the drug in BRAFV600E melanoma cells. These observations were analogous to how Escherichia coli encountering cell stress and nutrient deprivation can up-regulate and activate DinB/pol IV, the bacterial ortholog of Polκ, to induce mutagenesis that enables stress tolerance or escape. However, we found that the increased expression of Polκ was not excessively mutagenic, indicating that noncatalytic or other functions of Polκ could mediate its role in stress responses in mammalian cells. Repressing the expression or nuclear localization of Polκ might prevent drug resistance in some cancer cells.

Regenerative lineages and immune-mediated pruning in lung cancer metastasis.

Developmental processes underlying normal tissue regeneration have been implicated in cancer, but the degree of their enactment during tumor progression and under the selective pressures of immune surveillance, remain unknown. Here we show that human primary lung adenocarcinomas are characterized by the emergence of regenerative cell types, typically seen in response to lung injury, and by striking infidelity among transcription factors specifying most alveolar and bronchial epithelial lineages. In contrast, metastases are enriched for key endoderm and lung-specifying transcription factors, SOX2 and SOX9, and recapitulate more primitive transcriptional programs spanning stem-like to regenerative pulmonary epithelial progenitor states. This developmental continuum mirrors the progressive stages of spontaneous outbreak from metastatic dormancy in a mouse model and exhibits SOX9-dependent resistance to natural killer cells. Loss of developmental stage-specific constraint in macrometastases triggered by natural killer cell depletion suggests a dynamic interplay between developmental plasticity and immune-mediated pruning during metastasis.

Cancer modeling by Transgene Electroporation in Adult Zebrafish (TEAZ).

Transgenic animals are invaluable for modeling cancer genomics, but often require complex crosses of multiple germline alleles to obtain the desired combinations. Zebrafish models have advantages in that transgenes can be rapidly tested by mosaic expression, but typically lack spatial and temporal control of tumor onset, which limits their utility for the study of tumor progression and metastasis. To overcome these limitations, we have developed a method referred to as Transgene Electroporation in Adult Zebrafish (TEAZ). TEAZ can deliver DNA constructs with promoter elements of interest to drive fluorophores, oncogenes or CRISPR-Cas9-based mutagenic cassettes in specific cell types. Using TEAZ, we created a highly aggressive melanoma model via Cas9-mediated inactivation of Rb1 in the context of BRAFV600E in spatially constrained melanocytes. Unlike prior models that take ∼4 months to develop, we found that TEAZ leads to tumor onset in ∼7 weeks, and these tumors develop in fully immunocompetent animals. As the resulting tumors initiated at highly defined locations, we could track their progression via fluorescence, and documented deep invasion into tissues and metastatic deposits. TEAZ can be deployed to other tissues and cell types, such as the heart, with the use of suitable transgenic promoters. The versatility of TEAZ makes it widely accessible for rapid modeling of somatic gene alterations and cancer progression at a scale not achievable in other in vivo systems.

Adipocyte-Derived Lipids Mediate Melanoma Progression via FATP Proteins.

Advanced, metastatic melanomas frequently grow in subcutaneous tissues and portend a poor prognosis. Though subcutaneous tissues are largely composed of adipocytes, the mechanisms by which adipocytes influence melanoma are poorly understood. Using in vitro and in vivo models, we find that adipocytes increase proliferation and invasion of adjacent melanoma cells. Additionally, adipocytes directly transfer lipids to melanoma cells, which alters tumor cell metabolism. Adipocyte-derived lipids are transferred to melanoma cells through the FATP/SLC27A family of lipid transporters expressed on the tumor cell surface. Among the six FATP/SLC27A family members, melanomas significantly overexpress FATP1/SLC27A1. Melanocyte-specific FATP1 expression cooperates with BRAFV600E in transgenic zebrafish to accelerate melanoma development, an effect that is similarly seen in mouse xenograft studies. Pharmacologic blockade of FATPs with the small-molecule inhibitor Lipofermata abrogates lipid transport into melanoma cells and reduces melanoma growth and invasion. These data demonstrate that stromal adipocytes can drive melanoma progression through FATP lipid transporters and represent a new target aimed at interrupting adipocyte-melanoma cross-talk.Significance: We demonstrate that stromal adipocytes are donors of lipids that mediate melanoma progression. Adipocyte-derived lipids are taken up by FATP proteins that are aberrantly expressed in melanoma. Inhibition of FATPs decreases melanoma lipid uptake, invasion, and growth. We provide a mechanism for how stromal adipocytes drive tumor progression and demonstrate a novel microenvironmental therapeutic target. Cancer Discov; 8(8); 1006-25. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 899.

Distant Insulin Signaling Regulates Vertebrate Pigmentation through the Sheddase Bace2.

Patterning of vertebrate melanophores is essential for mate selection and protection from UV-induced damage. Patterning can be influenced by circulating long-range factors, such as hormones, but it is unclear how their activity is controlled in recipient cells to prevent excesses in cell number and migration. The zebrafish wanderlust mutant harbors a mutation in the sheddase bace2 and exhibits hyperdendritic and hyperproliferative melanophores that localize to aberrant sites. We performed a chemical screen to identify suppressors of the wanderlust phenotype and found that inhibition of insulin/PI3Kγ/mTOR signaling rescues the defect. In normal physiology, Bace2 cleaves the insulin receptor, whereas its loss results in hyperactive insulin/PI3K/mTOR signaling. Insulin B, an isoform enriched in the head, drives the melanophore defect. These results suggest that insulin signaling is negatively regulated by melanophore-specific expression of a sheddase, highlighting how long-distance factors can be regulated in a cell-type-specific manner.

A Quantitative System for Studying Metastasis Using Transparent Zebrafish.

Metastasis is the defining feature of advanced malignancy, yet remains challenging to study in laboratory environments. Here, we describe a high-throughput zebrafish system for comprehensive, in vivo assessment of metastatic biology. First, we generated several stable cell lines from melanomas of transgenic mitfa-BRAF(V600E);p53(-/-) fish. We then transplanted the melanoma cells into the transparent casper strain to enable highly quantitative measurement of the metastatic process at single-cell resolution. Using computational image analysis of the resulting metastases, we generated a metastasis score, µ, that can be applied to quantitative comparison of metastatic capacity between experimental conditions. Furthermore, image analysis also provided estimates of the frequency of metastasis-initiating cells (∼1/120,000 cells). Finally, we determined that the degree of pigmentation is a key feature defining cells with metastatic capability. The small size and rapid generation of progeny combined with superior imaging tools make zebrafish ideal for unbiased high-throughput investigations of cell-intrinsic or microenvironmental modifiers of metastasis. The approaches described here are readily applicable to other tumor types and thus serve to complement studies also employing murine and human cell culture systems.

MicroRNA 223 is upregulated in the multistep progression of Barrett's esophagus and modulates sensitivity to chemotherapy by targeting PARP1.

PURPOSE: Recent microarray and RNA-sequencing studies have uncovered aberrantly expressed microRNAs (miRNA) in Barrett's esophagus-associated esophageal adenocarcinoma. The functional significance of these miRNAs in esophageal adenocarcinoma initiation and progression is largely unknown. EXPERIMENTAL DESIGN: Expression levels of miR-199a/b-3p, -199a-5p, -199b-5p, -200b, -200c, -223, and -375 were determined in microdissected tissues from cardiac mucosa, Barrett's esophagus, dysplastic Barrett's esophagus, and esophageal adenocarcinoma using quantitative real-time PCR. miR-223 expression was validated in precursors and esophageal adenocarcinomas from 95 patients with esophageal adenocarcinoma by in situ hybridization (ISH). miR-223 was transfected into two esophageal adenocarcinoma cell lines, and in vitro assays were conducted. Target genes were identified using Illumina microarray, and results were validated in cell lines and human specimens. RESULTS: miR-199 family members and miR-223 were significantly overexpressed in esophageal adenocarcinoma, however, only miR-223 showed a stepwise increase during esophageal adenocarcinoma carcinogenesis. A similar trend was observed by ISH, which additionally showed that miR-223 is exclusively expressed by the epithelial compartment. miR-223-overexpressing cells had statistically significantly more migratory and invasive potential than scramble sequence-transfected cells. PARP1 was identified as a direct target gene of miR-223 in esophageal adenocarcinoma cells. Increased sensitivity to chemotherapy was observed in cells with enforced miR-223 expression and reduced PARP1. CONCLUSIONS: miR-223 is significantly upregulated during the Barrett's esophagus-dysplasia-esophageal adenocarcinoma sequence. Although high miR-223 levels might contribute to an aggressive phenotype, our results also suggest that patients with esophageal adenocarcinoma with high miR-223 levels might benefit from treatment with DNA-damaging agents.

Notch signaling pathway targeted therapy suppresses tumor progression and metastatic spread in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) remains a lethal human malignancy with historically limited success in treatment. The role of aberrant Notch signaling, which requires the constitutive activation of γ-secretase, in the initiation and progression of PDA is well defined and inhibitors of this pathway are currently in clinical trials. Here we investigated the in vivo therapeutic effect of PF-03084014, a selective γ-secretase inhibitor, alone and in combination with gemcitabine in pancreatic cancer xenografts. PF-03084014 treatment inhibited the cleavage of nuclear Notch 1 intracellular domain and Notch targets Hes-1 and Hey-1. Gemcitabine treatment showed good response but not capable of inducing tumor regressions and targeting the tumor-resident cancer stem cells (CD24(+)CD44(+) and ALDH(+) tumor cells). A combination of PF-03084014 and gemcitabine treatment resulted tumor regression in 3 of 4 subcutaneously implanted xenograft models. PF-03084014, and in combination with gemcitabine reduced putative cancer stem cells, indicating that PF-03084014 target the especially dangerous and resilient cancer stem cells within pancreatic tumors. Tumor re-growth curves plotted after drug treatments demonstrated that the effect of the combination therapy was sustainable than that of gemcitabine. Notably, in a highly aggressive orthotopic model, PF-03084014 and gemcitabine combination was effective in inducing apoptosis, inhibition of tumor cell proliferation and angiogenesis, resulting in the attenuation of primary tumor growth as well as controlling metastatic dissemination, compared to gemcitabine treatment. In summary, our preclinical data suggest that PF-03084014 has greater anti-tumor activity in combination with gemcitabine in PDA and provides rationale for further investigation of this combination in PDA.

Coordinated effects of microRNA-494 induce G2/M arrest in human cholangiocarcinoma.

MicroRNA (miRs) have emerged as salient regulators in cancer homeostasis and, recently, as putative therapeutics. Cholangiocarcinomas (CCA) are aggressive cancers with survival usually measured in months. mRNA arrays followed by pathway analysis revealed that miR-494 is a major modulator of the cell cycle progression from gap 2 (G2) to mitosis (M). We performed fluorescence activated cell sorting (FACS) as well as differential interference contrast (DIC) microscopy, and confirmed that miR-494 induces a significant arrest in G2/M in CCA cells. Furthermore, we verified that miR-494 modulates the protein level of six genes involved in the G2/M transition: Polo-like Kinase 1 (PLK1), pituitary tumor-transforming gene 1 (PTTG1), Cyclin B1 (CCNB1), cell-division cycle 2 (CDC2), cell-division cycle 20 (CDC20) and topoisomerase II α (TOP2A). Next, we identified direct binding of miR-494 to the open reading frame (ORF) and downregulation of PTTG1 and TOP2A. In summary, our findings suggest that miR-494 has a global regulatory role in cell cycle progression, exerted by concerted effects on multiple proteins involved in gap 1 (G1) to synthesis (S), as described previously, as well as G2 to M progression. Therefore, it appears that the simultaneous effects of a single miR species on multiple targets along the same canonical pathway is advantageous for the usage of miRs as therapeutics. In addition, our data suggest that miRs act within a narrow range. miR expression above the upper threshold does not appear to induce further effects, which is reassuring in terms of off-target effects of miR surrounding noncancerous tissue.

A composite polymer nanoparticle overcomes multidrug resistance and ameliorates doxorubicin-associated cardiomyopathy.

Acquired chemotherapy resistance is a major contributor to treatment failure in oncology. For example, the efficacy of the common anticancer agent doxorubicin (DOX) is limited by the emergence of multidrug resistance (MDR) phenotype in cancer cells. While dose escalation of DOX can circumvent such resistance to a degree, this is precluded by the appearance of cardiotoxicity, a particularly debilitating condition in children. In vitro studies have established the ability of the natural phytochemical curcumin to overcome MDR; however, its widespread clinical application is restricted by poor solubility and low bioavailability. Building upon our recently developed polymer nanoparticle of curcumin (NanoCurc or NC) that significantly enhances the systemic bioavailability of curcumin, we synthesized a doxorubicin-curcumin composite nanoparticle formulation called NanoDoxCurc (NDC) for overcoming DOX resistance. Compared to DOX alone, NDC inhibited the MDR phenotype and caused striking growth inhibition both in vitro and in vivo in several models of DOX-resistant cancers (multiple myeloma, acute leukemia, prostate and ovarian cancers, respectively). Notably, NDC-treated mice also demonstrated complete absence of cardiac toxicity, as assessed by echocardiography, or any bone marrow suppression, even at cumulative dosages where free DOX and pegylated liposomal DOX (Doxil®) resulted in demonstrable attenuation of cardiac function and hematological toxicities. This improvement in safety profile was achieved through a reduction of DOX-induced intracellular oxidative stress, as indicated by total glutathione levels and glutathione peroxidase activity in cardiac tissue. A composite DOX-curcumin nanoparticle that overcomes both MDR-based DOX chemoresistance and DOX-induced cardiotoxicity holds promise for providing lasting and safe anticancer therapy.

The gamma secretase inhibitor MRK-003 attenuates pancreatic cancer growth in preclinical models.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy, with most patients facing an adverse clinical outcome. Aberrant Notch pathway activation has been implicated in the initiation and progression of PDAC, specifically the aggressive phenotype of the disease. We used a panel of human PDAC cell lines as well as patient-derived PDAC xenografts to determine whether pharmacologic targeting of Notch pathway could inhibit PDAC growth and potentiate gemcitabine sensitivity. MRK-003, a potent and selective γ-secretase inhibitor, treatment resulted in the downregulation of nuclear Notch1 intracellular domain, inhibition of anchorage-independent growth, and reduction of tumor-initiating cells capable of extensive self-renewal. Pretreatment of PDAC cells with MRK-003 in cell culture significantly inhibited the subsequent engraftment in immunocompromised mice. MRK-003 monotherapy significantly blocked tumor growth in 5 of 9 (56%) PDAC xenografts. A combination of MRK-003 and gemcitabine showed enhanced antitumor effects compared with gemcitabine in 4 of 9 (44%) PDAC xenografts, reduced tumor cell proliferation, and induced both apoptosis and intratumoral necrosis. Gene expression analysis of untreated tumors indicated that upregulation of NF-κB pathway components was predictive of sensitivity to MRK-003, whereas upregulation in B-cell receptor signaling and nuclear factor erythroid-derived 2-like 2 pathway correlated with response to the combination of MRK-003 with gemcitabine. Our findings strengthen the rationale for small-molecule inhibition of Notch signaling as a therapeutic strategy in PDAC.

Mucin 16 (cancer antigen 125) expression in human tissues and cell lines and correlation with clinical outcome in adenocarcinomas of the pancreas, esophagus, stomach, and colon.

Mucin 16 (cancer antigen 125) is a cell surface glycoprotein that plays a role in promoting cancer cell growth in ovarian cancer. The aims of this study were to examine mucin 16 expression in a large number of digestive tract adenocarcinomas and precursors and to determine whether mucin 16 up-regulation is correlated with patient outcome. Tissue microarrays were constructed using surgical resection tissues and included pancreatic (115 normal, 29 precursors, 200 pancreatic ductal adenocarcinomas), esophageal (86 normal, 104 precursors, 95 esophageal adenocarcinomas, 35 lymph node metastases), gastric (211 normal, 8 precursors, 119 gastric adenocarcinomas, 62 lymph node metastases), and colorectal (34 normal, 17 precursors, 39 colorectal adenocarcinomas) tissues. Mucin 16 was detected in 81.5%, 69.9%, 41.2%, and 64.1% of the pancreatic ductal adenocarcinomas, esophageal adenocarcinomas, gastric adenocarcinomas, and colorectal adenocarcinomas, respectively. Mucin 16 was seen in a subset of the precursors. On multivariate analysis, moderate/diffuse mucin 16 in pancreatic ductal adenocarcinomas was strongly associated with poor survival (P < .001), independent of other prognosis predictors. A similar trend was observed for esophageal adenocarcinomas (P = .160) and gastric adenocarcinomas (P = .080). Focal mucin 16 in colorectal adenocarcinomas was significantly correlated (P = .044) with a better patient outcome, when compared with mucin 16-negative cases. Using Western blot analysis, we found mucin 16 expression in 3 of 6 pancreatic ductal adenocarcinoma and 1 of 2 esophageal adenocarcinoma cell lines. We conclude that most of the digestive tract adenocarcinomas and a subset of their precursors express mucin 16. Mucin 16 expression is an independent predictor of poor outcome in pancreatic ductal adenocarcinomas and potentially in esophageal adenocarcinomas and gastric adenocarcinomas. We propose that mucin 16 may function as a prognostic marker and therapeutic target in the future.

A polymeric nanoparticle encapsulated small-molecule inhibitor of Hedgehog signaling (NanoHHI) bypasses secondary mutational resistance to Smoothened antagonists.

Aberrant activation of the hedgehog (Hh) signaling pathway is one of the most prevalent abnormalities in human cancer. Tumors with cell autonomous Hh activation (e.g., medulloblastomas) can acquire secondary mutations at the Smoothened (Smo) antagonist binding pocket, which render them refractory to conventional Hh inhibitors. A class of Hh pathway inhibitors (HPI) has been identified that block signaling downstream of Smo; one of these compounds, HPI-1, is a potent antagonist of the Hh transcription factor Gli1 and functions independent of upstream components in the pathway. Systemic administration of HPI-1 is challenging due to its minimal aqueous solubility and poor bioavailability. We engineered a polymeric nanoparticle from [poly(lactic-co-glycolic acid); (PLGA)] conjugated with polyethylene glycol (PEG), encapsulating HPI-1 (NanoHHI). NanoHHI particles have an average diameter of approximately 60 nm, forms uniform aqueous suspension, and improved systemic bioavailability compared with the parent compound. In contrast to the prototype targeted Smo antagonist, HhAntag (Genentech), NanoHHI markedly inhibits the growth of allografts derived from Ptch(-/+); Trp53(-/-) mouse medulloblastomas that harbor a Smo(D477G) binding site mutation (P < 0.001), which is accompanied by significant downregulation of mGli1 as well as bona fide Hh target genes (Akna, Cltb, and Olig2). Notably, NanoHHI combined with gemcitabine also significantly impedes the growth of orthotopic Pa03C pancreatic cancer xenografts that have a ligand-dependent, paracrine mechanism of Hh activation when compared with gemcitabine alone. No demonstrable hematologic or biochemical abnormalities were observed with NanoHHI administration. NanoHHI should be amenable to clinical translation in settings where tumors acquire mutational resistance to current Smo antagonists.

Molecular determinants of retinoic acid sensitivity in pancreatic cancer.

PURPOSE: To identify a predictive molecular "signature" for sensitivity to retinoic acid in pancreatic cancer. EXPERIMENTAL DESIGN: Fourteen patient-derived, low-passage pancreatic ductal adenocarcinoma (PDAC) lines with varied expression of fatty acid-binding protein 5 (FABP5) and cellular retinoic acid-binding protein 2 (CRABP2) were used to evaluate the response to all-trans retinoic acid (ATRA). Cell proliferation, apoptosis, and migration/invasion assays were used to measure the in vitro response. Tumor growth was monitored in subcutaneous xenografts in athymic nude mice for 4 weeks. RESULTS: Response to ATRA was observed to be dependent upon differential expression of FABP5 versus CRABP2. Thus, elevated FABP5 expression was associated with minimal cytotoxicity and tumor growth inhibition and a paradoxical increase in migration and invasion. Conversely, CRABP2 expression in the absence of FABP5 was associated with significant tumor growth inhibition with ATRA, even in gemcitabine-resistant tumors. The ATRA-resistant phenotype of FABP5(high)CRABP2(null) cells could be circumvented by ectopic expression of CRABP2. Alternatively, reexpression of endogenous CRABP2 could be enabled in FABP5(high)CRABP2(null) PDAC lines by exposure to decitabine and trichostatin A, thereby relieving epigenetic silencing of the CRABP2 gene promoter. Immunohistochemical staining for FABP5 in archival human tissue microarrays identifies a subset of cases (13 of 63, ~20%) which are negative for FABP5 expression and might be candidates for ATRA therapy. CONCLUSIONS: The widely used agent ATRA deserves a "second look" in PDAC, but needs to be targeted to patient subsets with biopsy-proven FABP5-negative tumors, or be combined with a chromatin-modifying agent to reexpress endogenous CRABP2.

Restitution of tumor suppressor microRNAs using a systemic nanovector inhibits pancreatic cancer growth in mice.

Mis-expression of microRNAs (miRNA) is widespread in human cancers, including in pancreatic cancer. Aberrations of miRNA include overexpression of oncogenic miRs (Onco-miRs) or downregulation of so-called tumor suppressor TSG-miRs. Restitution of TSG-miRs in cancer cells through systemic delivery is a promising avenue for pancreatic cancer therapy. We have synthesized a lipid-based nanoparticle for systemic delivery of miRNA expression vectors to cancer cells (nanovector). The plasmid DNA-complexed nanovector is approximately 100 nm in diameter and shows no apparent histopathologic or biochemical evidence of toxicity upon intravenous injection. Two miRNA candidates known to be downregulated in the majority of pancreatic cancers were selected for nanovector delivery: miR-34a, which is a component of the p53 transcriptional network and regulates cancer stem cell survival, and the miR-143/145 cluster, which together repress the expression of KRAS2 and its downstream effector Ras-responsive element binding protein-1 (RREB1). Systemic intravenous delivery with either miR-34a or miR-143/145 nanovectors inhibited the growth of MiaPaCa-2 subcutaneous xenografts (P < 0.01 for miR-34a; P < 0.05 for miR-143/145); the effects were even more pronounced in the orthotopic (intrapancreatic) setting (P < 0.0005 for either nanovector) when compared with vehicle or mock nanovector delivering an empty plasmid. Tumor growth inhibition was accompanied by increased apoptosis and decreased proliferation. The miRNA restitution was confirmed in treated xenografts by significant upregulation of the corresponding miRNA and significant decreases in specific miRNA targets (SIRT1, CD44 and aldehyde dehydrogenase for miR34a, and KRAS2 and RREB1 for miR-143/145). The nanovector is a platform with potential broad applicability in systemic miRNA delivery to cancer cells.

On-demand drug delivery from self-assembled nanofibrous gels: a new approach for treatment of proteolytic disease.

Local delivery of drugs offers the potential for high local drug concentration while minimizing systemic toxicity, which is often observed with oral dosing. However, local depots are typically administered less frequently and include an initial burst followed by a continuous release. To maximize efficiency of therapy, it is critical to ensure that drug is only released when needed. One of the hallmarks of rheumatoid arthritis, for example, is its variable disease activity consisting of exacerbations of inflammation punctuated by periods of remission. This presents significant challenges for matching localized drug delivery with disease activity. An optimal system would be nontoxic and only release drugs during the period of exacerbation, self-titrating in response to the level of inflammation. We report the development of an injectable self-assembled nanofibrous hydrogel, from a generally recognized as safe material, which is capable of encapsulation and release of agents in response to specific enzymes that are significantly upregulated in a diseased state including matrix metalloproteinases (MMP-2 and MMP-9) and esterases. We show that these self-assembled nanofibrous gels can withstand shear forces that may be experienced in dynamic environments such as joints, can remain stable following injection into healthy joints of mice, and can disassemble in vitro to release encapsulated agents in response to synovial fluid from arthritic patients. This novel approach represents a next-generation therapeutic strategy for localized treatment of proteolytic diseases.