Yellow emission carbon dots for highly selective and sensitive OFF-ON sensing of ferric and pyrophosphate ions in living cells.

A simple "OFF-ON" fluorescent system was proposed for selective and sensitive detection of ferric ion (Fe3+) and pyrophosphate (PPi) in living cells. The method was constructed based on the bright yellow emission of carbon dots (y-CDs), which were prepared using o-phenylenediamine (OPD) as the precursor via a facile hydrothermal treatment. The as-obtained y-CDs, with an average size of 2.6 nm, exhibited an excitation-independent emission peak at 574 nm. The fluorescence of y-CDs can be remarkably quenched by Fe3+ with high selectivity and sensitivity. Interestingly, the quenched fluorescence can be recovered regularly upon addition of PPi, showing a promising detection for PPi. The linear ranges for Fe3+ and PPi detections were 0.05-80 and 0.5-120 µM, respectively, and the corresponding limit of detections (LODs) were 22.1 and 73.9 nM. As we proved the y-CDs have negligible cytotoxicity and excellent biocompatibility, further application to the fluorescence imaging of intracellular Fe3+ and PPi were conducted, suggesting the prepared y-CDs can be used to monitor Fe3+ and PPi variation in living cells. Overall, our developed y-CDs-based OFF-ON switch fluorescent probe has the advantages of simplicity, agility, high sensitivity and selectivity, which provides a promising platform for environmental and biology applications, and paves a new avenue for monitoring the hydrolysis process of adenosine triphosphate disodium salt (ATP) by detection of PPi in organisms.

Lineage-specific control of TFIIH by MITF determines transcriptional homeostasis and DNA repair.

The melanocytic lineage, which is prominently exposed to ultraviolet radiation (UVR) and radiation-independent oxidative damage, requires specific DNA-damage response mechanisms to maintain genomic and transcriptional homeostasis. The coordinate lineage-specific regulation of intricately intertwined DNA repair and transcription is incompletely understood. Here we demonstrate that the Microphthalmia-associated transcription factor (MITF) directly controls general transcription and UVR-induced nucleotide excision repair by transactivation of GTF2H1 as a core element of TFIIH. Thus, MITF ensures the rapid resumption of transcription after completion of strand repair and maintains transcriptional output, which is indispensable for survival of the melanocytic lineage including melanoma in vitro and in vivo. Moreover, MITF controls c-MYC implicated in general transcription by transactivation of far upstream binding protein 2 (FUBP2/KSHRP), which induces c-MYC pulse regulation through TFIIH, and experimental depletion of MITF results in consecutive loss of CDK7 in the TFIIH-CAK subcomplex. Targeted for proteasomal degradation, CDK7 is dependent on transactivation by MITF or c-MYC to maintain a steady state. The dependence of TFIIH-CAK on sequence-specific MITF and c-MYC constitutes a previously unrecognized mechanism feeding into super-enhancer-driven or other oncogenic transcriptional circuitries, which supports the concept of a transcription-directed therapeutic intervention in melanoma.

Analysis of Single-Cell RNA-Seq Identifies Cell-Cell Communication Associated with Tumor Characteristics.

Tumor ecosystems are composed of multiple cell types that communicate by ligand-receptor interactions. Targeting ligand-receptor interactions (for instance, with immune checkpoint inhibitors) can provide significant benefits for patients. However, our knowledge of which interactions occur in a tumor and how these interactions affect outcome is still limited. We present an approach to characterize communication by ligand-receptor interactions across all cell types in a microenvironment using single-cell RNA sequencing. We apply this approach to identify and compare the ligand-receptor interactions present in six syngeneic mouse tumor models. To identify interactions potentially associated with outcome, we regress interactions against phenotypic measurements of tumor growth rate. In addition, we quantify ligand-receptor interactions between T cell subsets and their relation to immune infiltration using a publicly available human melanoma dataset. Overall, this approach provides a tool for studying cell-cell interactions, their variability across tumors, and their relationship to outcome.

Autophosphorylation of the carboxyl-terminal domain is not required for oncogenic transformation by lung-cancer derived EGFR mutants.

Aberrant activation of cancer-derived mutants of the epidermal growth factor receptor (EGFR) is closely associated with cancer pathogenesis and is thought to be mediated through multiple tyrosine phosphorylations within the C-terminal domain. Here, we examined the consequences of the loss of these C-terminal phosphorylation sites on cellular transformation in the context of lung-cancer-derived L858R, exon 19 deletion and exon 20 insertion mutant EGFR. Oncogenic EGFR mutants with substitution of the 10 potential C-terminal tyrosine autophosphorylation sites for phenylalanine (CYF10) were still able to promote anchorage-independent growth in soft agar at levels comparable to the parental L858R or exon19 deletion or exon 20 insertion mutants with intact autophosphorylation sites. Furthermore, these CYF10 mutants retained the ability to transform Ba/F3 cells in the absence of IL-3. Bead-based phosphorylation and immunoprecipitation analyses demonstrated that key EGFR-associated proteins-including Grb2 and PLC-γ-are neither phosphorylated nor bound to CYF10 mutants in transformed cells. Taken together, we conclude that tyrosine phosphorylation is not required for oncogenic activity of lung-cancer-derived mutant EGFR, suggesting these mutants can lead to cellular transformation by an alternative mechanism independent of EGFR phosphorylation.

Estimation of immune cell content in tumour tissue using single-cell RNA-seq data.

As interactions between the immune system and tumour cells are governed by a complex network of cell-cell interactions, knowing the specific immune cell composition of a solid tumour may be essential to predict a patient's response to immunotherapy. Here, we analyse in depth how to derive the cellular composition of a solid tumour from bulk gene expression data by mathematical deconvolution, using indication-specific and cell type-specific reference gene expression profiles (RGEPs) from tumour-derived single-cell RNA sequencing data. We demonstrate that tumour-derived RGEPs are essential for the successful deconvolution and that RGEPs from peripheral blood are insufficient. We distinguish nine major cell types, as well as three T cell subtypes. Using the tumour-derived RGEPs, we can estimate the content of many tumours associated immune and stromal cell types, their therapeutically relevant ratios, as well as an improved gene expression profile of the malignant cells.

HER2+ Cancer Cell Dependence on PI3K vs. MAPK Signaling Axes Is Determined by Expression of EGFR, ERBB3 and CDKN1B.

Understanding the molecular pathways by which oncogenes drive cancerous cell growth, and how dependence on such pathways varies between tumors could be highly valuable for the design of anti-cancer treatment strategies. In this work we study how dependence upon the canonical PI3K and MAPK cascades varies across HER2+ cancers, and define biomarkers predictive of pathway dependencies. A panel of 18 HER2+ (ERBB2-amplified) cell lines representing a variety of indications was used to characterize the functional and molecular diversity within this oncogene-defined cancer. PI3K and MAPK-pathway dependencies were quantified by measuring in vitro cell growth responses to combinations of AKT (MK2206) and MEK (GSK1120212; trametinib) inhibitors, in the presence and absence of the ERBB3 ligand heregulin (NRG1). A combination of three protein measurements comprising the receptors EGFR, ERBB3 (HER3), and the cyclin-dependent kinase inhibitor p27 (CDKN1B) was found to accurately predict dependence on PI3K/AKT vs. MAPK/ERK signaling axes. Notably, this multivariate classifier outperformed the more intuitive and clinically employed metrics, such as expression of phospho-AKT and phospho-ERK, and PI3K pathway mutations (PIK3CA, PTEN, and PIK3R1). In both cell lines and primary patient samples, we observed consistent expression patterns of these biomarkers varies by cancer indication, such that ERBB3 and CDKN1B expression are relatively high in breast tumors while EGFR expression is relatively high in other indications. The predictability of the three protein biomarkers for differentiating PI3K/AKT vs. MAPK dependence in HER2+ cancers was confirmed using external datasets (Project Achilles and GDSC), again out-performing clinically used genetic markers. Measurement of this minimal set of three protein biomarkers could thus inform treatment, and predict mechanisms of drug resistance in HER2+ cancers. More generally, our results show a single oncogenic transformation can have differing effects on cell signaling and growth, contingent upon the molecular and cellular context.

A novel role for microphthalmia-associated transcription factor-regulated pigment epithelium-derived factor during melanoma progression.

Microphthalmia-associated transcription factor (MITF) acts via pigment epithelium-derived factor (PEDF), an antiangiogenic protein, to regulate retinal pigment epithelium migration. PEDF expression and/or regulation during melanoma development have not been investigated previously. Using immunohistochemistry, we determined expression of PEDF in common and dysplastic melanocytic nevi, melanoma in situ, invasive melanoma, and metastatic melanoma (n = 102). PEDF expression was consistently decreased in invasive and metastatic melanoma, compared with nevi and melanoma in situ (P < 0.0001). PEDF was lost in thicker melanomas (P = 0.003), and correlated with depth of invasion (P = 0.003) and distant metastasis (P = 0.0331), but only marginally with mitotic index, AJCC stage, nodal metastasis, or blood vascular density (0.05 < P < 0.10). Quantitative real-time PCR and microarray analyses confirmed PEDF down-regulation at the mRNA level in several melanoma lines, compared with melanocytes. MITF positively correlated with PEDF expression in invasive melanomas (P = 0.0003). Searching for PEDF regulatory mechanisms revealed two occupied conserved E-boxes (DNA recognition elements) in the first intron of the human and mouse PEDF promoter regions, confirmed by binding assays. Dominant-negative and siRNA approaches in vivo demonstrated direct transcriptional influence of MITF on PEDF, establishing the PEDF gene (SERPINF1) as a MITF target in melanocytes and melanoma cells. These findings suggest that loss of PEDF expression promotes early invasive melanoma growth.

SYK is a critical regulator of FLT3 in acute myeloid leukemia.

Cooperative dependencies between mutant oncoproteins and wild-type proteins are critical in cancer pathogenesis and therapy resistance. Although spleen tyrosine kinase (SYK) has been implicated in hematologic malignancies, it is rarely mutated. We used kinase activity profiling to identify collaborators of SYK in acute myeloid leukemia (AML) and determined that FMS-like tyrosine kinase 3 (FLT3) is transactivated by SYK via direct binding. Highly activated SYK is predominantly found in FLT3-ITD positive AML and cooperates with FLT3-ITD to activate MYC transcriptional programs. FLT3-ITD AML cells are more vulnerable to SYK suppression than FLT3 wild-type counterparts. In a FLT3-ITD in vivo model, SYK is indispensable for myeloproliferative disease (MPD) development, and SYK overexpression promotes overt transformation to AML and resistance to FLT3-ITD-targeted therapy.

High-throughput tyrosine kinase activity profiling identifies FAK as a candidate therapeutic target in Ewing sarcoma.

Limited progress has been made in the treatment of advanced-stage pediatric solid tumors despite the accelerated pace of cancer discovery over the last decade. Tyrosine kinase inhibition is one tractable therapeutic modality for treating human malignancy. However, little is known about the kinases critical to the development or maintenance of many pediatric solid tumors such as Ewing sarcoma. Using a fluorescent, bead-based technology to profile activated tyrosine kinases, we identified focal adhesion kinase (FAK, PTK2) as a candidate target in Ewing sarcoma. FAK is a tyrosine kinase critical for cellular adhesion, growth, and survival. As such, it is a compelling target for cancer-based therapy. In this study, we have shown that FAK is highly phosphorylated in primary Ewing sarcoma tumor samples and that downregulation of FAK by short hairpin RNA and treatment with a FAK-selective kinase inhibitor, PF-562271, impaired growth and colony formation in Ewing sarcoma cell lines. Moreover, treatment of Ewing sarcoma cell lines with PF-562271 induced apoptosis and led to downregulation of AKT/mTOR and CAS activity. Finally, we showed that small-molecule inhibition of FAK attenuated Ewing sarcoma tumor growth in vivo. With FAK inhibitors currently in early-phase clinical trials for adult malignancies, these findings may bear immediate relevance to patients with Ewing sarcoma.

TSC1 involvement in bladder cancer: diverse effects and therapeutic implications.

TSC1 is often mutated in bladder cancer. However the importance of this event in disease pathogenesis and its implications for therapy are uncertain. We used genomic sequencing to examine the involvement of TSC1 in bladder cancer, and signalling pathway analysis and small-molecule screening to identify targeted therapeutic strategies in TSC1 mutant bladder cancer cell lines. TSC1 loss of heterozygosity was seen in 54% of bladder cancers. Two (4.9%) of these 41 bladder cancers had TSC1 mutations by exon-based sequencing. Analysis of 27 bladder cancer cell lines demonstrated inactivating TSC1 mutations in three: RT-4, HCV29, 97-1. Interestingly, only RT-4 showed classic feedback inhibition of AKT, and was highly sensitive to treatment with mTOR inhibitors rapamycin and Torin1. 97-1 cells showed constitutive EGFR activation, and were highly sensitive to combined treatment with the mTOR inhibitor Torin1 and EGFR inhibitors Lapatinib or Afatinib. A BRAF missense mutation G469V was found in HCV29 cells, and AKT activation was dependent on BRAF, but independent of ERK. A kinase inhibitor screen of HCV29 cells showed strong growth inhibition by the Hsp90 inhibitor NVP-AUY922, and we then found synergistic inhibitory effects of NVP-AUY922 combined with either Torin1 or rapamycin on cell survival for both HCV29 and 97-1 cells. In aggregate, these findings indicate that there are highly variable mutation profiles and signalling pathway activation in TSC1-mutant bladder cancer. Furthermore, combined Hsp90/mTOR inhibition is a promising therapeutic approach for TSC1 mutant bladder cancer.

Molecular dissection of AKT activation in lung cancer cell lines.

AKT is a critical signaling node downstream of phosphoinositide 3-kinase (PI3K), which is often activated in cancer. We analyzed the state of activation of AKT in 80 human non-small cell lung carcinoma cell lines under serum starvation conditions. We identified 13 lines, which showed persistent AKT activation in the absence of serum. In 12 of 13 lines, AKT activation could be attributed to loss of PTEN, activating mutation in EGF receptor (EGFR) or PIK3CA, or amplification of ERBB2. HCC2429 was the only cell line that had no alterations in those genes, but had high phospho-AKT(Ser473) levels under serum starvation conditions. However, the activation of AKT in HCC2429 was PI3K- and mTOR complex 2 (mTORC2)-dependent based upon use of specific inhibitors. Kinome tyrosine phosphorylation profiling showed that both Notch and SRC were highly activated in this cell line. Despite the activation of Notch, AKT activation and cell survival were not affected by Notch inhibitors DAPT or compound E. In contrast, SRC inhibitors PP2 and dasatinib both significantly decreased pAKT(Ser473) levels and reduced cell survival by inducing apoptosis. Furthermore, a combination of SRC and mTOR inhibition synergistically blocked activation of AKT and induced apoptosis. Overexpression of SRC has been identified previously in human lung cancers, and these results suggest that a combination of SRC and mTOR inhibitors may have unique therapeutic benefit for a subset of lung cancers with these molecular features.

Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion.

Drug resistance presents a challenge to the treatment of cancer patients. Many studies have focused on cell-autonomous mechanisms of drug resistance. By contrast, we proposed that the tumour micro-environment confers innate resistance to therapy. Here we developed a co-culture system to systematically assay the ability of 23 stromal cell types to influence the innate resistance of 45 cancer cell lines to 35 anticancer drugs. We found that stroma-mediated resistance is common, particularly to targeted agents. We characterized further the stroma-mediated resistance of BRAF-mutant melanoma to RAF inhibitors because most patients with this type of cancer show some degree of innate resistance. Proteomic analysis showed that stromal cell secretion of hepatocyte growth factor (HGF) resulted in activation of the HGF receptor MET, reactivation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-OH kinase (PI(3)K)-AKT signalling pathways, and immediate resistance to RAF inhibition. Immunohistochemistry experiments confirmed stromal cell expression of HGF in patients with BRAF-mutant melanoma and showed a significant correlation between HGF expression by stromal cells and innate resistance to RAF inhibitor treatment. Dual inhibition of RAF and either HGF or MET resulted in reversal of drug resistance, suggesting RAF plus HGF or MET inhibitory combination therapy as a potential therapeutic strategy for BRAF-mutant melanoma. A similar resistance mechanism was uncovered in a subset of BRAF-mutant colorectal and glioblastoma cell lines. More generally, this study indicates that the systematic dissection of interactions between tumours and their micro-environment can uncover important mechanisms underlying drug resistance.

Global tyrosine kinome profiling of human thyroid tumors identifies Src as a promising target for invasive cancers.

BACKGROUND: Novel therapies are needed for the treatment of invasive thyroid cancers. Aberrant activation of tyrosine kinases plays an important role in thyroid oncogenesis. Because current targeted therapies are biased toward a small subset of tyrosine kinases, we conducted a study to reveal novel therapeutic targets for thyroid cancer using a bead-based, high-throughput system. METHODS: Thyroid tumors and matched normal tissues were harvested from twenty-six patients in the operating room. Protein lysates were analyzed using the Luminex immunosandwich, a bead-based kinase phosphorylation assay. Data was analyzed using GenePattern 3.0 software and clustered according to histology, demographic factors, and tumor status regarding capsular invasion, size, lymphovascular invasion, and extrathyroidal extension. Survival and invasion assays were performed to determine the effect of Src inhibition in papillary thyroid cancer (PTC) cells. RESULTS: Tyrosine kinome profiling demonstrated upregulation of nine tyrosine kinases in tumors relative to matched normal thyroid tissue: EGFR, PTK6, BTK, HCK, ABL1, TNK1, GRB2, ERK, and SRC. Supervised clustering of well-differentiated tumors by histology, gender, age, or size did not reveal significant differences in tyrosine kinase activity. However, supervised clustering by the presence of invasive disease showed increased Src activity in invasive tumors relative to non-invasive tumors (60% v. 0%, p<0.05). In vitro, we found that Src inhibition in PTC cells decreased cell invasion and proliferation. CONCLUSION: Global kinome analysis enables the discovery of novel targets for thyroid cancer therapy. Further investigation of Src targeted therapy for advanced thyroid cancer is warranted.

A novel ALK secondary mutation and EGFR signaling cause resistance to ALK kinase inhibitors.

Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKI), including crizotinib, are effective treatments in preclinical models and in cancer patients with ALK-translocated cancers. However, their efficacy will ultimately be limited by the development of acquired drug resistance. Here we report two mechanisms of ALK TKI resistance identified from a crizotinib-treated non-small cell lung cancer (NSCLC) patient and in a cell line generated from the resistant tumor (DFCI076) as well as from studying a resistant version of the ALK TKI (TAE684)-sensitive H3122 cell line. The crizotinib-resistant DFCI076 cell line harbored a unique L1152R ALK secondary mutation and was also resistant to the structurally unrelated ALK TKI TAE684. Although the DFCI076 cell line was still partially dependent on ALK for survival, it also contained concurrent coactivation of epidermal growth factor receptor (EGFR) signaling. In contrast, the TAE684-resistant (TR3) H3122 cell line did not contain an ALK secondary mutation but instead harbored coactivation of EGFR signaling. Dual inhibition of both ALK and EGFR was the most effective therapeutic strategy for the DFCI076 and H3122 TR3 cell lines. We further identified a subset (3/50; 6%) of treatment naive NSCLC patients with ALK rearrangements that also had concurrent EGFR activating mutations. Our studies identify resistance mechanisms to ALK TKIs mediated by both ALK and by a bypass signaling pathway mediated by EGFR. These mechanisms can occur independently, or in the same cancer, suggesting that the combination of both ALK and EGFR inhibitors may represent an effective therapy for these subsets of NSCLC patients.

Amplification of CRKL induces transformation and epidermal growth factor receptor inhibitor resistance in human non-small cell lung cancers.

UNLABELLED: We previously identified a region of recurrent amplification on chromosome 22q11.21 in a subset of primary lung adenocarcinomas. Here we show that CRKL, encoding for an adaptor protein, is amplified and overexpressed in non-small cell lung cancer (NSCLC) cells that harbor 22q11.21 amplifications. Overexpression of CRKL in immortalized human airway epithelial cells promoted anchorage-independent growth and tumorigenicity. Oncogenic CRKL activates the SOS1-RAS-RAF-ERK and SRC-C3G-RAP1 pathways. Suppression of CRKL in NSCLC cells that harbor CRKL amplifications induced cell death. Overexpression of CRKL in epidermal growth factor receptor (EGFR)-mutant cells induces resistance to gefitinib by activating extracellular signal-regulated kinase and AKT signaling. We identified CRKL amplification in an EGFR inhibitor-treated lung adenocarcinoma that was not present before treatment. These observations demonstrate that CRKL overexpression induces cell transformation, credential CRKL as a therapeutic target for a subset of NSCLC that harbor CRKL amplifications, and implicate CRKL as an additional mechanism of resistance to EGFR-directed therapy. SIGNIFICANCE: These studies credential CRKL as an oncogene in a subset of NSCLC. Overexpression of CRKL induces cell transformation and resistance to epidermal growth factor receptor inhibitor treatment and suggest that therapeutic interventions targeting CRKL may confer a clinical benefit in a defined subset of NSCLCs.

Mitogen-inducible gene-6 is a multifunctional adaptor protein with tumor suppressor-like activity in papillary thyroid cancer.

CONTEXT: Low tumoral expression of mitogen-inducible gene-6 (Mig-6) is associated with papillary thyroid cancer (PTC) recurrence after thyroidectomy. OBJECTIVE: We hypothesize that Mig-6 behaves as a tumor suppressor in PTC. DESIGN: Mig-6 expression and promoter methylation status were compared in 31 PTC specimens with matched normal thyroid tissue from the same patient. The impact of Mig-6 loss and gain of function on nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) activation, global tyrosine kinase phosphorylation, and cellular invasion was determined in vitro. RESULTS: Mig-6 protein was abundant in all normal thyroid specimens, whereas 77% of PTC had low Mig-6 expression. Mig-6 promoter methylation was found in 79% of PTC with low Mig-6 expression. Low Mig-6 expression in PTC specimens was associated with low NF-κB activity but high levels of epidermal growth factor receptor (EGFR) and ERK phosphorylation. Mig-6 expression inversely correlated with PTC size but had no association with other clinicopathological variables including age, extrathyroidal extension, lymphovascular invasion, or histological subtype. Mig-6 knockdown in thyroid cancer cell lines resulted in EGFR phosphorylation and diminished NF-κB activity, whereas Mig-6 overexpression had the opposite effects. Mig-6 knockdown activated ErbB2, Met, and Src phosphorylation. Furthermore, Mig-6 regulated ERK phosphorylation independent from its effects on EGFR. Mig-6 knockdown promoted cellular proliferation, as determined by clonogenic survival. Lastly, Mig-6 knockdown increased matrix metalloproteinase-2 and -9 activities and increased cellular invasion. CONCLUSIONS: Mig-6 has tumor suppressor-like activity in PTC. In vivo studies are required to confirm that Mig-6 is a putative tumor suppressor in PTC, and future studies should investigate the utility of Mig-6 as a diagnostic marker.

Autophagy induction with RAD001 enhances chemosensitivity and radiosensitivity through Met inhibition in papillary thyroid cancer.

Although autophagy is generally considered a prosurvival mechanism that preserves viability, there is evidence that it could drive an alternative programmed cell death pathway in cells with defects in apoptosis. Because the inhibition of autophagic activity promotes resistance to both chemotherapy and external beam radiation in papillary thyroid cancer (PTC), we determined if RAD001, a potent activator of autophagy, improves the efficacy of either therapy. We found that RAD001 increased the expression level of light chain 3-II, a marker for autophagy, as well as autophagosome formation in cell lines and in human PTC ex vivo. RAD001 sensitized PTC to doxorubicin and external beam radiation in a synergistic fashion, suggesting that combination therapy could improve therapeutic response at less toxic concentrations. The effects of RAD001 were abrogated by RNAi knockdown of the autophagy-related gene 5, suggesting that RAD001 acts, in part, by enhancing autophagy. Because the synergistic activity of RAD001 with doxorubicin and external radiation suggests distinct and complementary mechanisms of action, we characterized how autophagy modulates signaling pathways in PTC. To do so, we performed kinome profiling and discovered that autophagic activation resulted in Src phosphorylation and Met dephosphorylation. Src inhibition did not reverse the effects of RAD001, whereas Met inhibition reversed the effects of autophagy blockade on chemosensitivity. These results suggest that the anticancer effects of autophagic activation are mediated largely through Met. We conclude that RAD001 induces autophagy, which enhances the therapeutic response to cytotoxic chemotherapy and external beam radiation in PTC.

Key roles for transforming growth factor beta in melanocyte stem cell maintenance.

Melanocyte stem cells in the bulge area of hair follicles are responsible for hair pigmentation, and defects in them cause hair graying. Here we describe the process of melanocyte stem cell entry into the quiescent state and show that niche-derived transforming growth factor beta (TGF-beta) signaling plays important roles in this process. In vitro, TGF-beta not only induces reversible cell cycle arrest, but also promotes melanocyte immaturity by downregulating MITF, the master transcriptional regulator of melanocyte differentiation, and its downstream melanogenic genes. In vivo, TGF-beta signaling is activated in melanocyte stem cells when they reenter the quiescent noncycling state during the hair cycle and this process requires Bcl2 for cell survival. Furthermore, targeted TGF-beta type II receptor (TGFbRII) deficiency in the melanocyte lineage causes incomplete maintenance of melanocyte stem cell immaturity and results in mild hair graying. These data demonstrate that the TGF-beta signaling pathway is one of the key niche factors that regulate melanocyte stem cell immaturity and quiescence.

Bead-based profiling of tyrosine kinase phosphorylation identifies SRC as a potential target for glioblastoma therapy.

The aberrant activation of tyrosine kinases represents an important oncogenic mechanism, and yet the majority of such events remain undiscovered. Here we describe a bead-based method for detecting phosphorylation of both wild-type and mutant tyrosine kinases in a multiplexed, high-throughput and low-cost manner. With the aim of establishing a tyrosine kinase-activation catalog, we used this method to profile 130 human cancer lines. Follow-up experiments on the finding that SRC is frequently phosphorylated in glioblastoma cell lines showed that SRC is also activated in primary glioblastoma patient samples and that the SRC inhibitor dasatinib (Sprycel) inhibits viability and cell migration in vitro and tumor growth in vivo. Testing of dasatinib-resistant tyrosine kinase alleles confirmed that SRC is indeed the relevant target of dasatinib, which inhibits many tyrosine kinases. These studies establish the feasibility of tyrosine kinome-wide phosphorylation profiling and point to SRC as a possible therapeutic target in glioblastoma.

Assessing the significance of chromosomal aberrations in cancer: methodology and application to glioma.

Comprehensive knowledge of the genomic alterations that underlie cancer is a critical foundation for diagnostics, prognostics, and targeted therapeutics. Systematic efforts to analyze cancer genomes are underway, but the analysis is hampered by the lack of a statistical framework to distinguish meaningful events from random background aberrations. Here we describe a systematic method, called Genomic Identification of Significant Targets in Cancer (GISTIC), designed for analyzing chromosomal aberrations in cancer. We use it to study chromosomal aberrations in 141 gliomas and compare the results with two prior studies. Traditional methods highlight hundreds of altered regions with little concordance between studies. The new approach reveals a highly concordant picture involving approximately 35 significant events, including 16-18 broad events near chromosome-arm size and 16-21 focal events. Approximately half of these events correspond to known cancer-related genes, only some of which have been previously tied to glioma. We also show that superimposed broad and focal events may have different biological consequences. Specifically, gliomas with broad amplification of chromosome 7 have properties different from those with overlapping focalEGFR amplification: the broad events act in part through effects on MET and its ligand HGF and correlate with MET dependence in vitro. Our results support the feasibility and utility of systematic characterization of the cancer genome.