Zinc Alpha-2-Glycoprotein (ZAG/AZGP1) secreted by triple-negative breast cancer promotes tumor microenvironment fibrosis.

Obesity is a predisposition factor for breast cancer, suggesting a localized, reciprocal interaction between breast cancer cells and the surrounding mammary white adipose tissue. To investigate how breast cancer cells alter the composition and function of adipose tissue, we screened the secretomes of ten human breast cancer cell lines for the ability to modulate the differentiation of adipocyte stem and progenitor cells (ASPC). The screen identified a key adipogenic modulator, Zinc Alpha-2-Glycoprotein (ZAG/AZGP1), secreted by triple-negative breast cancer (TNBC) cells. TNBC-secreted ZAG inhibits adipogenesis and instead induces the expression of fibrotic genes. Accordingly, depletion of ZAG in TNBC cells attenuates fibrosis in white adipose tissue and inhibits tumor growth. Further, high expression of ZAG in TNBC patients, but not other clinical subtypes of breast cancer, is linked to poor prognosis. Our findings suggest a role of TNBC-secreted ZAG in promoting the transdifferentiation of ASPCs into cancer-associated fibroblasts to support tumorigenesis.

Genetic Silencing of AKT Induces Melanoma Cell Death via mTOR Suppression.

Aberrant activation of the PI3K-AKT pathway is common in many cancers, including melanoma, and AKT1, 2 and 3 (AKT1-3) are bona fide oncoprotein kinases with well-validated downstream effectors. However, efforts to pharmacologically inhibit AKT have proven to be largely ineffective. In this study, we observed paradoxical effects following either pharmacologic or genetic inhibition of AKT1-3 in melanoma cells. Although pharmacological inhibition was without effect, genetic silencing of all three AKT paralogs significantly induced melanoma cell death through effects on mTOR. This phenotype was rescued by exogenous AKT1 expression in a kinase-dependent manner. Pharmacological inhibition of PI3K and mTOR with a novel dual inhibitor effectively suppressed melanoma cell proliferation in vitro and inhibited tumor growth in vivo. Furthermore, this single-agent-targeted therapy was well-tolerated in vivo and was effective against MAPK inhibitor-resistant patient-derived melanoma xenografts. These results suggest that inhibition of PI3K and mTOR with this novel dual inhibitor may represent a promising therapeutic strategy in this disease in both the first-line and MAPK inhibitor-resistant setting.

Combining the Tyrosine Kinase Inhibitor Cabozantinib and the mTORC1/2 Inhibitor Sapanisertib Blocks ERK Pathway Activity and Suppresses Tumor Growth in Renal Cell Carcinoma.

Current treatment approaches for renal cell carcinoma (RCC) face challenges in achieving durable tumor responses due to tumor heterogeneity and drug resistance. Combination therapies that leverage tumor molecular profiles could offer an avenue for enhancing treatment efficacy and addressing the limitations of current therapies. To identify effective strategies for treating RCC, we selected ten drugs guided by tumor biology to test in six RCC patient-derived xenograft (PDX) models. The multitargeted tyrosine kinase inhibitor (TKI) cabozantinib and mTORC1/2 inhibitor sapanisertib emerged as the most effective drugs, particularly when combined. The combination demonstrated favorable tolerability and inhibited tumor growth or induced tumor regression in all models, including two from patients who experienced treatment failure with FDA-approved TKI and immunotherapy combinations. In cabozantinib-treated samples, imaging analysis revealed a significant reduction in vascular density, and single-nucleus RNA sequencing (snRNA-seq) analysis indicated a decreased proportion of endothelial cells in the tumors. SnRNA-seq data further identified a tumor subpopulation enriched with cell-cycle activity that exhibited heightened sensitivity to the cabozantinib and sapanisertib combination. Conversely, activation of the epithelial-mesenchymal transition pathway, detected at the protein level, was associated with drug resistance in residual tumors following combination treatment. The combination effectively restrained ERK phosphorylation and reduced expression of ERK downstream transcription factors and their target genes implicated in cell-cycle control and apoptosis. This study highlights the potential of the cabozantinib plus sapanisertib combination as a promising treatment approach for patients with RCC, particularly those whose tumors progressed on immune checkpoint inhibitors and other TKIs. SIGNIFICANCE: The molecular-guided therapeutic strategy of combining cabozantinib and sapanisertib restrains ERK activity to effectively suppress growth of renal cell carcinomas, including those unresponsive to immune checkpoint inhibitors.

Allele-Specific Gene Regulation, Phenotypes, and Therapeutic Vulnerabilities in Estrogen Receptor Alpha-Mutant Endometrial Cancer.

Activating estrogen receptor alpha (ER; also known as ESR1) mutations are present in primary endometrial and metastatic breast cancers, promoting estrogen-independent activation of the receptor. Functional characterizations in breast cancer have established unique molecular and phenotypic consequences of the receptor, yet the impact of ER mutations in endometrial cancer has not been fully explored. In this study, we used CRISPR-Cas9 to model the clinically prevalent ER-Y537S mutation and compared results with ER-D538G to discover allele-specific differences between ER mutations in endometrial cancer. We found that constitutive activity of mutant ER resulted in changes in the expression of thousands of genes, stemming from combined alterations to ER binding and chromatin accessibility. The unique gene expression programs resulted in ER-mutant cells developing increased cancer-associated phenotypes, including migration, invasion, anchorage-independent growth, and growth in vivo. To uncover potential treatment strategies, we identified ER-associated proteins via Rapid Immunoprecipitation and Mass Spectrometry of Endogenous Proteins and interrogated two candidates, CDK9 and NCOA3. Inhibition of these regulatory proteins resulted in decreased growth and migration, representing potential novel treatment strategies for ER-mutant endometrial cancer. IMPLICATIONS: This study provides insight into mutant ER activity in endometrial cancer and identifies potential therapies for women with ER-mutant endometrial cancer.

Characterization of HCI-EC-23 a novel estrogen- and progesterone-responsive endometrial cancer cell line.

Most endometrial cancers express the hormone receptor estrogen receptor alpha (ER) and are driven by excess estrogen signaling. However, evaluation of the estrogen response in endometrial cancer cells has been limited by the availability of hormonally responsive in vitro models, with one cell line, Ishikawa, being used in most studies. Here, we describe a novel, adherent endometrioid endometrial cancer (EEC) cell line model, HCI-EC-23. We show that HCI-EC-23 retains ER expression and that ER functionally responds to estrogen induction over a range of passages. We also demonstrate that this cell line retains paradoxical activation of ER by tamoxifen, which is also observed in Ishikawa and is consistent with clinical data. The mutational landscape shows that HCI-EC-23 is mutated at many of the commonly altered genes in EEC, has relatively few copy-number alterations, and is microsatellite instable high (MSI-high). In vitro proliferation of HCI-EC-23 is strongly reduced upon combination estrogen and progesterone treatment. HCI-EC-23 exhibits strong estrogen dependence for tumor growth in vivo and tumor size is reduced by combination estrogen and progesterone treatment. Molecular characterization of estrogen induction in HCI-EC-23 revealed hundreds of estrogen-responsive genes that significantly overlapped with those regulated in Ishikawa. Analysis of ER genome binding identified similar patterns in HCI-EC-23 and Ishikawa, although ER exhibited more bound sites in Ishikawa. This study demonstrates that HCI-EC-23 is an estrogen- and progesterone-responsive cell line model that can be used to study the hormonal aspects of endometrial cancer.

A human breast cancer-derived xenograft and organoid platform for drug discovery and precision oncology.

Models that recapitulate the complexity of human tumors are urgently needed to develop more effective cancer therapies. We report a bank of human patient-derived xenografts (PDXs) and matched organoid cultures from tumors that represent the greatest unmet need: endocrine-resistant, treatment-refractory and metastatic breast cancers. We leverage matched PDXs and PDX-derived organoids (PDxO) for drug screening that is feasible and cost-effective with in vivo validation. Moreover, we demonstrate the feasibility of using these models for precision oncology in real time with clinical care in a case of triple-negative breast cancer (TNBC) with early metastatic recurrence. Our results uncovered a Food and Drug Administration (FDA)-approved drug with high efficacy against the models. Treatment with this therapy resulted in a complete response for the individual and a progression-free survival (PFS) period more than three times longer than their previous therapies. This work provides valuable methods and resources for functional precision medicine and drug development for human breast cancer.

BRAFV600E induces reversible mitotic arrest in human melanocytes via microrna-mediated suppression of AURKB.

Benign melanocytic nevi frequently emerge when an acquired BRAFV600E mutation triggers unchecked proliferation and subsequent arrest in melanocytes. Recent observations have challenged the role of oncogene-induced senescence in melanocytic nevus formation, necessitating investigations into alternative mechanisms for the establishment and maintenance of proliferation arrest in nevi. We compared the transcriptomes of melanocytes from healthy human skin, nevi, and melanomas arising from nevi and identified a set of microRNAs as highly expressed nevus-enriched transcripts. Two of these microRNAs-MIR211-5p and MIR328-3p-induced mitotic failure, genome duplication, and proliferation arrest in human melanocytes through convergent targeting of AURKB. We demonstrate that BRAFV600E induces a similar proliferation arrest in primary human melanocytes that is both reversible and conditional. Specifically, BRAFV600E expression stimulates either arrest or proliferation depending on the differentiation state of the melanocyte. We report genome duplication in human melanocytic nevi, reciprocal expression of AURKB and microRNAs in nevi and melanomas, and rescue of arrested human nevus cells with AURKB expression. Taken together, our data describe an alternative molecular mechanism for melanocytic nevus formation that is congruent with both experimental and clinical observations.

Lots of people have small dark patches on their skin known as moles. Most moles form when individual cells known as melanocytes in the skin acquire a specific genetic mutation in a gene called BRAF. This mutation causes the cells to divide rapidly to form the mole. After a while, most moles stop growing and remain harmless for the rest of a person's life. Melanoma is a type of skin cancer that develops from damaged melanocytes. The same mutation in BRAF that is found in moles is also present in half of all cases of melanoma. Unlike in moles, the melanoma-causing mutation makes the melanocytes divide rapidly to form a tumor that keeps on growing indefinitely. It remains unclear why the same genetic mutation in the BRAF gene has such different consequences in moles and melanomas. To address this question, McNeal et al. used genetic approaches to study melanocytes from moles and melanomas. The experiments identified some molecules known as microRNAs that are present at higher levels in moles than in melanomas. Increasing the levels of two of these microRNAs in melanocytes from human skin stopped the cells from growing and dividing by inhibiting a gene called AURKB. This suggested that these microRNAs are responsible for halting the growth of moles. Introducing the mutated form of BRAF into melanocytes also stopped cells from growing and dividing by inhibiting AURKB. However, changing the environment surrounding the cells reversed this effect and allowed the melanocytes to resume dividing. In this way the mutated form of BRAF acts like a switch that allows melanocytes in skin cancers to start growing again under certain conditions. Further experiments found that a drug called barasertib is able to inhibit the growth of melanoma cells with the mutant form of BRAF. Future work will investigate whether it is possible to use this drug and other tools to stop skin cancer tumors from growing, and possibly even prevent skin tumors from forming in the first place.

Aspirin Protects Melanocytes and Keratinocytes against UVB-Induced DNA Damage In Vivo.

UVR promotes skin cancer through multiple mechanisms, including induction of inflammation, oxidative stress, and DNA damage such as 8-oxoguanine and cyclobutane pyrimidine dimers. We investigated whether the anti-inflammatory activities of aspirin (acetylsalicylic acid [ASA]) could protect against UVB-induced DNA damage and skin carcinogenesis. ASA reduced UVB-induced 8-oxoguanine and cyclobutane pyrimidine dimers in Melan-A melanocytes and HaCaT keratinocytes. Skin from UVB-irradiated C57BL/6 mice receiving 0.4 mg ASA daily by gavage exhibited less inflammation, fewer sunburn cells, and reduced 8-oxoguanine lesions than skin from irradiated control animals. ASA similarly reduced UVB-induced sunburn cells, 8-oxoguanine, and cyclobutane pyrimidine dimer lesions in skin of melanoma-prone TN61R mice, and this was associated with decreased prostaglandin E2 in plasma and skin. These effects of ASA, however, did not delay melanoma onset in TN61R mice exposed to a single neonatal dose of UVB. In SKH1-E mice prone to squamous cell carcinoma, ASA reduced plasma and skin prostaglandin E2 levels and indices of UVB-induced DNA damage and delayed squamous cell carcinoma onset induced by chronic UVB. These results indicate that ASA can protect against UVB-induced inflammation in skin and reduce UVB-induced DNA damage in both melanocytes and keratinocytes. These effects translated into greater chemopreventive efficacy for UVB-induced squamous cell carcinoma than melanoma mouse models.

Epigenetic silencing of TGFBI confers resistance to trastuzumab in human breast cancer.

BACKGROUND: Acquired resistance to trastuzumab is a major clinical problem in the treatment of HER2-positive (HER2+) breast cancer patients. The selection of trastuzumab-resistant patients is a great challenge of precision oncology. The aim of this study was to identify novel epigenetic biomarkers associated to trastuzumab resistance in HER2+ BC patients. METHODS: We performed a genome-wide DNA methylation (450K array) and a transcriptomic analysis (RNA-Seq) comparing trastuzumab-sensitive (SK) and trastuzumab-resistant (SKTR) HER2+ human breast cancer cell models. The methylation and expression levels of candidate genes were validated by bisulfite pyrosequencing and qRT-PCR, respectively. Functional assays were conducted in the SK and SKTR models by gene silencing and overexpression. Methylation analysis in 24 HER2+ human BC samples with complete response or non-response to trastuzumab-based treatment was conducted by bisulfite pyrosequencing. RESULTS: Epigenomic and transcriptomic analysis revealed the consistent hypermethylation and downregulation of TGFBI, CXCL2, and SLC38A1 genes in association with trastuzumab resistance. The DNA methylation and expression levels of these genes were validated in both sensitive and resistant models analyzed. Of the genes, TGFBI presented the highest hypermethylation-associated silencing both at the transcriptional and protein level. Ectopic expression of TGFBI in the SKTR model suggest an increased sensitivity to trastuzumab treatment. In primary tumors, TGFBI hypermethylation was significantly associated with trastuzumab resistance in HER2+ breast cancer patients. CONCLUSIONS: Our results suggest for the first time an association between the epigenetic silencing of TGFBI by DNA methylation and trastuzumab resistance in HER2+ cell models. These results provide the basis for further clinical studies to validate the hypermethylation of TGFBI promoter as a biomarker of trastuzumab resistance in HER2+ breast cancer patients.

MYC-Driven Small-Cell Lung Cancer is Metabolically Distinct and Vulnerable to Arginine Depletion.

PURPOSE: Small-cell lung cancer (SCLC) has been treated clinically as a homogeneous disease, but recent discoveries suggest that SCLC is heterogeneous. Whether metabolic differences exist among SCLC subtypes is largely unexplored. In this study, we aimed to determine whether metabolic vulnerabilities exist between SCLC subtypes that can be therapeutically exploited. EXPERIMENTAL DESIGN: We performed steady state metabolomics on tumors isolated from distinct genetically engineered mouse models (GEMM) representing the MYC- and MYCL-driven subtypes of SCLC. Using genetic and pharmacologic approaches, we validated our findings in chemo-naïve and -resistant human SCLC cell lines, multiple GEMMs, four human cell line xenografts, and four newly derived PDX models. RESULTS: We discover that SCLC subtypes driven by different MYC family members have distinct metabolic profiles. MYC-driven SCLC preferentially depends on arginine-regulated pathways including polyamine biosynthesis and mTOR pathway activation. Chemo-resistant SCLC cells exhibit increased MYC expression and similar metabolic liabilities as chemo-naïve MYC-driven cells. Arginine depletion with pegylated arginine deiminase (ADI-PEG 20) dramatically suppresses tumor growth and promotes survival of mice specifically with MYC-driven tumors, including in GEMMs, human cell line xenografts, and a patient-derived xenograft from a relapsed patient. Finally, ADI-PEG 20 is significantly more effective than the standard-of-care chemotherapy. CONCLUSIONS: These data identify metabolic heterogeneity within SCLC and suggest arginine deprivation as a subtype-specific therapeutic vulnerability for MYC-driven SCLC.

Protective autophagy elicited by RAF→MEK→ERK inhibition suggests a treatment strategy for RAS-driven cancers.

Pancreatic ductal adenocarcinoma (PDA) was responsible for ~ 44,000 deaths in the United States in 2018 and is the epitome of a recalcitrant cancer driven by a pharmacologically intractable oncoprotein, KRAS1-4. Downstream of KRAS, the RAF→MEK→ERK signaling pathway plays a central role in pancreatic carcinogenesis5. However, paradoxically, inhibition of this pathway has provided no clinical benefit to patients with PDA6. Here we show that inhibition of KRAS→RAF→MEK→ERK signaling elicits autophagy, a process of cellular recycling that protects PDA cells from the cytotoxic effects of KRAS pathway inhibition. Mechanistically, inhibition of MEK1/2 leads to activation of the LKB1→AMPK→ULK1 signaling axis, a key regulator of autophagy. Furthermore, combined inhibition of MEK1/2 plus autophagy displays synergistic anti-proliferative effects against PDA cell lines in vitro and promotes regression of xenografted patient-derived PDA tumors in mice. The observed effect of combination trametinib plus chloroquine was not restricted to PDA as other tumors, including patient-derived xenografts (PDX) of NRAS-mutated melanoma and BRAF-mutated colorectal cancer displayed similar responses. Finally, treatment of a patient with PDA with the combination of trametinib plus hydroxychloroquine resulted in a partial, but nonetheless striking disease response. These data suggest that this combination therapy may represent a novel strategy to target RAS-driven cancers.

Publisher Correction: Protective autophagy elicited by RAF→MEK→ERK inhibition suggests a treatment strategy for RAS-driven cancers.

In the version of this article initially published, the label over the bottom schematic in Fig. 1a was "pH > 5.0"; it should have been "pH < 5.0". Further, the original article misspelt the surname of Katrin P. Guillen as "Gullien". These errors have been corrected in the print, PDF and HTML versions of the article.

Aspirin Suppresses PGE2 and Activates AMP Kinase to Inhibit Melanoma Cell Motility, Pigmentation, and Selective Tumor Growth In Vivo.

There are conflicting epidemiologic data on whether chronic aspirin (ASA) use may reduce melanoma risk in humans. Potential anticancer effects of ASA may be mediated by its ability to suppress prostaglandin E2 (PGE2) production and activate 5'-adenosine monophosphate-activated protein kinase (AMPK). We investigated the inhibitory effects of ASA in a panel of melanoma and transformed melanocyte cell lines, and on tumor growth in a preclinical model. ASA and the COX-2 inhibitor celecoxib did not affect melanoma cell viability, but significantly reduced colony formation, cell motility, and pigmentation (melanin production) in vitro at concentrations of 1 mmol/L and 20 µmol/L, respectively. ASA-mediated inhibition of cell migration and pigmentation was rescued by exogenous PGE2 or Compound C, which inhibits AMPK activation. Levels of tyrosinase, MITF, and p-ERK were unaffected by ASA exposure. Following a single oral dose of 0.4 mg ASA to NOD/SCID mice, salicylate was detected in plasma and skin at 4 hours and PGE2 levels were reduced up to 24 hours. Some human melanoma tumors xenografted into NOD/SCID mice were sensitive to chronic daily ASA administration, exhibiting reduced growth and proliferation. ASA-treated mice bearing sensitive and resistant tumors exhibited both decreased PGE2 in plasma and tumors and increased phosphorylated AMPK in tumors. We conclude that ASA inhibits colony formation, cell motility, and pigmentation through suppression of PGE2 and activation of AMPK and reduces growth of some melanoma tumors in vivo This preclinical model could be used for further tumor and biomarker studies to support future melanoma chemoprevention trials in humans. Cancer Prev Res; 11(10); 629-42. ©2018 AACR.

A class of extracellular vesicles from breast cancer cells activates VEGF receptors and tumour angiogenesis.

Non-classical secretory vesicles, collectively referred to as extracellular vesicles (EVs), have been implicated in different aspects of cancer cell survival and metastasis. Here, we describe how a specific class of EVs, called microvesicles (MVs), activates VEGF receptors and tumour angiogenesis through a unique 90 kDa form of VEGF (VEGF90K). We show that VEGF90K is generated by the crosslinking of VEGF165, catalysed by the enzyme tissue transglutaminase, and associates with MVs through its interaction with the chaperone Hsp90. We further demonstrate that MV-associated VEGF90K has a weakened affinity for Bevacizumab, causing Bevacizumab to be ineffective in blocking MV-dependent VEGF receptor activation. However, treatment with an Hsp90 inhibitor releases VEGF90K from MVs, restoring the sensitivity of VEGF90K to Bevacizumab. These findings reveal a novel mechanism by which cancer cell-derived MVs influence the tumour microenvironment and highlight the importance of recognizing their unique properties when considering drug treatment strategies.

Plasma exosome microRNAs are indicative of breast cancer.

BACKGROUND: microRNAs are promising candidate breast cancer biomarkers due to their cancer-specific expression profiles. However, efforts to develop circulating breast cancer biomarkers are challenged by the heterogeneity of microRNAs in the blood. To overcome this challenge, we aimed to develop a molecular profile of microRNAs specifically secreted from breast cancer cells. Our first step towards this direction relates to capturing and analyzing the contents of exosomes, which are small secretory vesicles that selectively encapsulate microRNAs indicative of their cell of origin. To our knowledge, circulating exosome microRNAs have not been well-evaluated as biomarkers for breast cancer diagnosis or monitoring. METHODS: Exosomes were collected from the conditioned media of human breast cancer cell lines, mouse plasma of patient-derived orthotopic xenograft models (PDX), and human plasma samples. Exosomes were verified by electron microscopy, nanoparticle tracking analysis, and western blot. Cellular and exosome microRNAs from breast cancer cell lines were profiled by next-generation small RNA sequencing. Plasma exosome microRNA expression was analyzed by qRT-PCR analysis. RESULTS: Small RNA sequencing and qRT-PCR analysis showed that several microRNAs are selectively encapsulated or highly enriched in breast cancer exosomes. Importantly, the selectively enriched exosome microRNA, human miR-1246, was detected at significantly higher levels in exosomes isolated from PDX mouse plasma, indicating that tumor exosome microRNAs are released into the circulation and can serve as plasma biomarkers for breast cancer. This observation was extended to human plasma samples where miR-1246 and miR-21 were detected at significantly higher levels in the plasma exosomes of 16 patients with breast cancer as compared to the plasma exosomes of healthy control subjects. Receiver operating characteristic curve analysis indicated that the combination of plasma exosome miR-1246 and miR-21 is a better indicator of breast cancer than their individual levels. CONCLUSIONS: Our results demonstrate that certain microRNA species, such as miR-21 and miR-1246, are selectively enriched in human breast cancer exosomes and significantly elevated in the plasma of patients with breast cancer. These findings indicate a potential new strategy to selectively analyze plasma breast cancer microRNAs indicative of the presence of breast cancer.

Preclinical Evaluation of Fatty Acid Synthase and EGFR Inhibition in Triple-Negative Breast Cancer.

PURPOSE: Triple-negative breast cancer (TNBC) lacks an approved targeted therapy. Despite initial good response to chemotherapy, 30% of the patients relapse within 5 years after treatment. EGFR overexpression is a common marker in TNBC, and its expression has been correlated with poor outcome. Inhibition of fatty acid synthase (FASN) activity leads to apoptosis of human carcinoma cells overexpressing FASN. We tested the hypothesis that blocking FASN in combination with anti-EGFR signaling agents would be an effective antitumor strategy in sensitive and chemoresistant TNBC. EXPERIMENTAL DESIGN: Several TNBC cell lines and 29 primary tumors were included to determine whether FASN is a potential target in TNBC. Doxorubicin-resistant TNBC cell lines (231DXR and HCCDXR) have been developed and characterized in our laboratory. Cellular and molecular interactions of anti-FASN compounds (EGCG and C75) with cetuximab were analyzed. In vivo tumor growth inhibition was evaluated after cetuximab, EGCG, or the combination in TNBC orthoxenograft models. RESULTS: TNBC cell lines showed overexpression of FASN enzyme and its inhibition correlated to FASN levels. FASN staining was observed in all of the 29 TNBC tumor samples. In vitro, EGCG and C75 plus cetuximab showed strong synergism in sensitive and chemoresistant cells. In vivo, the combination of EGCG with cetuximab displayed strong antitumor activity against the sensitive and chemoresistant TNBC orthoxenografts, without signs of toxicity. CONCLUSIONS: Our results show that the simultaneous blockade of FASN and EGFR is effective in preclinical models of sensitive and chemoresistant TNBC. Clin Cancer Res; 22(18); 4687-97. ©2016 AACR.

Vaccine-derived mutation in motif D of poliovirus RNA-dependent RNA polymerase lowers nucleotide incorporation fidelity.

All viral RNA-dependent RNA polymerases (RdRps) have a conserved structural element termed motif D. Studies of the RdRp from poliovirus (PV) have shown that a conformational change of motif D leads to efficient and faithful nucleotide addition by bringing Lys-359 into the active site where it serves as a general acid. The RdRp of the Sabin I vaccine strain has Thr-362 changed to Ile. Such a drastic change so close to Lys-359 might alter RdRp function and contribute in some way to the attenuated phenotype of Sabin type I. Here we present our characterization of the T362I RdRp. We find that the T362I RdRp exhibits a mutator phenotype in biochemical experiments in vitro. Using NMR, we show that this change in nucleotide incorporation fidelity correlates with a change in the structural dynamics of motif D. A recombinant PV expressing the T362I RdRp exhibits normal growth properties in cell culture but expresses a mutator phenotype in cells. For example, the T362I-containing PV is more sensitive to the mutagenic activity of ribavirin than wild-type PV. Interestingly, the T362I change was sufficient to cause a statistically significant reduction in viral virulence. Collectively, these studies suggest that residues of motif D can be targeted when changes in nucleotide incorporation fidelity are desired. Given the observation that fidelity mutants can serve as vaccine candidates, it may be possible to use engineering of motif D for this purpose.

Overview of human primary tumorgraft models: comparisons with traditional oncology preclinical models and the clinical relevance and utility of primary tumorgrafts in basic and translational oncology research.

Laboratory models that accurately replicate human tumor initiation and characteristics are integral to advancing knowledge in cancer research. However, comparative studies between commonly employed laboratory models and human tumors have demonstrated that some models have molecular and genomic alterations dissimilar to the cancer type they attempt to replicate. In contrast, several recent comparative studies suggest that because patient-derived tumors grown in mice maintain many of the important characteristics of the original tumor, they represent an important tool for the development of new cancer therapeutics. Detailed in this overview are the advantages and disadvantages of the most commonly used cancer models for mechanistic and therapeutic research, with an emphasis on the advances made in the production and use of patient-derived tumorgrafts.

Motif D of viral RNA-dependent RNA polymerases determines efficiency and fidelity of nucleotide addition.

Fast, accurate nucleotide incorporation by polymerases facilitates expression and maintenance of genomes. Many polymerases use conformational dynamics of a conserved α helix to permit efficient nucleotide addition only when the correct nucleotide substrate is bound. This α helix is missing in structures of RNA-dependent RNA polymerases (RdRps) and RTs. Here, we use solution-state nuclear magnetic resonance to demonstrate that the conformation of conserved structural motif D of an RdRp is linked to the nature (correct versus incorrect) of the bound nucleotide and the protonation state of a conserved, motif-D lysine. Structural data also reveal the inability of motif D to achieve its optimal conformation after incorporation of an incorrect nucleotide. Functional data are consistent with the conformational change of motif D becoming rate limiting during and after nucleotide misincorporation. We conclude that motif D of RdRps and, by inference, RTs is the functional equivalent to the fidelity helix of other polymerases.

Heparan sulfate sulfatase SULF2 regulates PDGFRα signaling and growth in human and mouse malignant glioma.

Glioblastoma (GBM), a uniformly lethal brain cancer, is characterized by diffuse invasion and abnormal activation of multiple receptor tyrosine kinase (RTK) signaling pathways, presenting a major challenge to effective therapy. The activation of many RTK pathways is regulated by extracellular heparan sulfate proteoglycans (HSPG), suggesting these molecules may be effective targets in the tumor microenvironment. In this study, we demonstrated that the extracellular sulfatase, SULF2, an enzyme that regulates multiple HSPG-dependent RTK signaling pathways, was expressed in primary human GBM tumors and cell lines. Knockdown of SULF2 in human GBM cell lines and generation of gliomas from Sulf2(-/-) tumorigenic neurospheres resulted in decreased growth in vivo in mice. We found a striking SULF2 dependence in activity of PDGFRα, a major signaling pathway in GBM. Ablation of SULF2 resulted in decreased PDGFRα phosphorylation and decreased downstream MAPK signaling activity. Interestingly, in a survey of SULF2 levels in different subtypes of GBM, the proneural subtype, characterized by aberrations in PDGFRα, demonstrated the strongest SULF2 expression. Therefore, in addition to its potential as an upstream target for therapy of GBM, SULF2 may help identify a subset of GBMs that are more dependent on exogenous growth factor-mediated signaling. Our results suggest the bioavailability of growth factors from the microenvironment is a significant contributor to tumor growth in a major subset of human GBM.