3D clusters of somatic mutations in cancer reveal numerous rare mutations as functional targets

Many mutations in cancer are of unknown functional significance. Standard methods use statistically significant recurrence of mutations in tumor samples as an indicator of functional impact. We extend such analyses into the long tail of rare mutations by considering recurrence of mutations in clusters of spatially close residues in protein structures. Analyzing 10,000 tumor exomes, we identify more than 3000 rarely mutated residues in proteins as potentially functional and experimentally validate several in RAC1 and MAP2K1. These potential driver mutations (web resources: 3dhotspots.org and cBioPortal.org) can extend the scope of genomically informed clinical trials and of personalized choice of therapy. Electronic supplementary material The online version of this article (doi:10.1186/s13073-016-0393-x) contains supplementary material, which is available to authorized users

Signal transducer and activator of transcription 3 (Stat3) phosphorylation in primary breast cancer

<p><b>Copyright information:</b></p><p>Taken from "Stat3 is tyrosine-phosphorylated through the interleukin-6/glycoprotein 130/Janus kinase pathway in breast cancer"</p><p>http://breast-cancer-research.com/content/9/3/R32</p><p>Breast Cancer Research 2007;9(3):R32-R32.</p><p>Published online 25 May 2007</p><p>PMCID:PMC1929096.</p><p></p> Tissue microarrays of primary breast tumors (85) were analyzed for nuclear tyrosine-phosphorylated Stat3 (pStat3) by immunohistochemical analysis. Ten percent expressed high levels (3+), 36% moderate levels (+2), 23% low levels (+1), and 31% no detectable pStat3 (0)

Signal transducer and activator of transcription 3 (Stat3) phosphorylation is mediated through Janus kinases (Jaks) but not epidermal growth factor receptor (EGFR) or Src kinases

<p><b>Copyright information:</b></p><p>Taken from "Stat3 is tyrosine-phosphorylated through the interleukin-6/glycoprotein 130/Janus kinase pathway in breast cancer"</p><p>http://breast-cancer-research.com/content/9/3/R32</p><p>Breast Cancer Research 2007;9(3):R32-R32.</p><p>Published online 25 May 2007</p><p>PMCID:PMC1929096.</p><p></p> Radioimmunoprecipitation assay (RIPA) extracts (50 μg) isolated from MDA-MB-468 cells treated for 4 hours with dimethyl sulfoxide (DMSO), P6 1 μM, BMS 50 nM, and ZD 2 μM were analyzed for pEGFR, total EGFR, tyrosine-phosphorylated Stat3 (pStat3), Stat3, pSrc, Src, pJak2, and Jak2. Nuclear extracts (20 μg) isolated from MDA-MB-435 cells (upper panels) and RIPA extracts (bottom panels) treated as in and probed with the indicated antibodies. Nuclear extracts (20 μg) isolated from MDA-MB-231 cells (upper panels) and RIPA extracts (bottom panels) treated as in and probed with the indicated antibodies. Ten thousand cells (MCF10A, MCF7, MDA-MB-468, and MDA-MB-435) were plated into a 96-well culture dish and treated with DMSO control or P6 (1 μM) for 48 hours, and proliferation was measured using an MTT (3- [4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay

Immunohisotchemical analaysis of primary breast cancer specimens for interleukin-6 (IL-6) (upper panels) and tyrosine-phosphorylated signal transducer and activator of transcription 3 (pStat3) (bottom panels)

<p><b>Copyright information:</b></p><p>Taken from "Stat3 is tyrosine-phosphorylated through the interleukin-6/glycoprotein 130/Janus kinase pathway in breast cancer"</p><p>http://breast-cancer-research.com/content/9/3/R32</p><p>Breast Cancer Research 2007;9(3):R32-R32.</p><p>Published online 25 May 2007</p><p>PMCID:PMC1929096.</p><p></p> Examples of high pStat3 and IL-6 (left panels) as well as low pStat3 and IL-6 (right panels) are indicated

Signal transducer and activator of transcription 3 (Stat3) activation is through the interleukin-6/glycoprotein 130/Janus kinase (IL-6/gp130/Jak) pathway

<p><b>Copyright information:</b></p><p>Taken from "Stat3 is tyrosine-phosphorylated through the interleukin-6/glycoprotein 130/Janus kinase pathway in breast cancer"</p><p>http://breast-cancer-research.com/content/9/3/R32</p><p>Breast Cancer Research 2007;9(3):R32-R32.</p><p>Published online 25 May 2007</p><p>PMCID:PMC1929096.</p><p></p> Nuclear extracts isolated from MDA-MB-468 (468) cells treated with immunoglobulin G (IgG) (10 μg/ml), P6 (1 μM), BR3 (10 μg/ml), and α-IL-6 (1 μg/ml) for 16 hours were analyzed for tyrosine-phosphorylated Stat3 (pStat3) and total Stat3 protein. Nuclear extracts isolated from MDA-MB-231 (231) cells treated with IgG (10 μg/ml), P6 (1 μM), BR3 (10 μg/ml), and α-IL-6 (1 μg/ml) for 16 hours were analyzed for pStat3 and total Stat3. Nuclear extracts isolated from MDA-MB-435 (435) cells treated with IgG (10 μg/ml), P6 (1 μM), and BR3 (10 μg/ml) for 16 hours were analyzed for pStat3 and total Stat3. Nuclear extracts isolated from 4175 and 231 cells treated with IgG (10 μg/ml) and BR3 (10 μg/ml) for 16 hours were analyzed for pStat3 and total Stat3. Nuclear extracts isolated from MCF10A cells treated for 30 minutes with conditioned media (CM) from MDA-MB-468 cells after pretreatment for 4 hours with IgG control antibody, BR3 (5 μg/ml), α-IL-6 antibody (5 μg/ml), α-oncostatin M (OSM) antibody (10 μg/ml), and α-leukemia inhibitory factor (LIF) antibody (10 μg/ml) were analyzed for pStat3 and total Stat3

Mutations in the EGFR kinase domain mediate STAT3 activation via IL-6 production in human lung adenocarcinomas

Persistently activated or tyrosine-phosphorylated STAT3 (pSTAT3) is found in 50% of lung adenocarcinomas. pSTAT3 is found in primary adenocarcinomas and cell lines harboring somatic-activating mutations in the tyrosine kinase domain of EGFR. Treatment of cell lines with either an EGFR inhibitor or an src kinase inhibitor had no effect on pSTAT3 levels, whereas a pan-JAK inhibitor (P6) blocked activation of STAT3 and inhibited tumorigenesis. Cell lines expressing these persistently activated mutant EGFRs also produced high IL-6 levels, and blockade of the IL-6/gp130/JAK pathway led to a decrease in pSTAT3 levels. In addition, reduction of IL-6 levels by RNA interference led to a decrease in tumorigenesis. Introduction of persistently activated EGFR into immortalized breast epithelial cells led to tumorigenesis, IL-6 expression, and STAT3 activation, all of which could be inhibited with P6 or gp130 blockade. Furthermore, inhibition of EGFR activity in multiple cell lines partially blocked transcription of IL-6 and concurrently decreased production and release of IL-6. Finally, immunohistochemical analysis revealed a positive correlation between pSTAT3 and IL-6 positivity in primary lung adenocarcinomas. Therefore, mutant EGFR could activate the gp130/JAK/STAT3 pathway by means of IL-6 upregulation in primary human lung adenocarcinomas, making this pathway a potential target for cancer treatment

The IL-6/JAK/Stat3 Feed-Forward Loop Drives Tumorigenesis and Metastasis

We have investigated the importance of interleukin-6 (IL-6) in promoting tumor growth and metastasis. In human primary breast cancers, increased levels of IL-6 were found at the tumor leading edge and positively correlated with advanced stage, suggesting a mechanistic link between tumor cell production of IL-6 and invasion. In support of this hypothesis, we showed that the IL-6/Janus kinase (JAK)/signal transducer and activator of transcription 3 (Stat3) pathway drives tumor progression through the stroma and metastatic niche. Overexpression of IL-6 in tumor cell lines promoted myeloid cell recruitment, angiogenesis, and induced metastases. We demonstrated the therapeutic potential of interrupting this pathway with IL-6 receptor blockade or by inhibiting its downstream effectors JAK1/2 or Stat3. These clinically relevant interventions did not inhibit tumor cell proliferation in vitro but had profound effects in vivo on tumor progression, interfering broadly with tumor-supportive stromal functions, including angiogenesis, fibroblast infiltration, and myeloid suppressor cell recruitment in both the tumor and pre-metastatic niche. This study provides the first evidence for IL-6 expression at the leading edge of invasive human breast tumors and demonstrates mechanistically that IL-6/JAK/Stat3 signaling plays a critical and pharmacologically targetable role in orchestrating the composition of the tumor microenvironment that promotes growth, invasion, and metastasis