Stromal-interaction molecule 1 (STIM1) is an endoplasmic reticulum Ca(2+) storage sensor that promotes cell growth, migration and angiogenesis in breast and cervical cancers. Here we report that the microtubule-associated histone deacetylase 6 (HDAC6) differentially regulates activation of STIM1-mediated store-operated Ca(2+) entry (SOCE) between cervical cancer cells and normal cervical epithelial cells. Confocal microscopy of living cells indicated that microtubule integrity was necessary for STIM1 trafficking to the plasma membrane and interaction with Orai1, an essential pore subunit of SOCE. Cancer cells overexpressed both STIM1 and Orai1 compared with normal cervical epithelial cells. HDAC6 upregulation in cancer cells was accompanied by hypoacetylated α-tubulin. Tubastatin-A, a specific HDAC6 inhibitor, inhibited STIM1 translocation to plasma membrane and blocked SOCE activation in cancer cells but not normal epithelial cells. Genetic or pharmacological inhibition of HDAC6 blocked STIM1 membrane trafficking and downstream Ca(2+) influx, as evidenced by total internal reflection fluorescent images and intracellular Ca(2+) determination. In contrast, HDAC6 inhibition did not affect interactions between STIM1 and the microtubule plus-end-binding protein EB1. Analysis of surgical specimens confirmed that most cervical cancer tissues overexpressed STIM1 and Orai1, accompanied by hypo-acetylated α-tubulin. Together, our results identify HDAC6 as a candidate target to disrupt STIM1-mediated SOCE as a general strategy to block malignant cell behavior.

Fanconi anemia (FA) is a genome-instability syndrome that has been associated with both cancer predisposition and bone-marrow failure. FA proteins are involved in cellular response to replication stress in which they coordinate DNA repair with DNA replication and cell cycle progression. One regulator of the replication stress response is the ATP-dependent DNA translocase FANCM which we have shown to be hyperphosphorylated in response to various genotoxic agents. However, the significance of this phosphorylation remained unclear. Here we show that genotoxic stress-induced FANCM phosphorylation is ATR dependent and that this modification is highly significant for the cellular response to replication stress. We identified serine (S1045) residue of FANCM that is phosphorylated in response to genotoxic stress and this effect is ATR dependent. We show that S1045 is required for FANCM functions including its role in FA pathway integrity, recruiting FANCM to the site of interstrand cross links, preventing the cells from entering mitosis prematurely, and efficient activation of the CHK1 and G2/M checkpoints. Overall, our data suggest that an ATR-FANCM feedback loop is present in the FA and replication-stress-response pathways, and that it is required for both efficient ATR/CHK1-checkpoint activation and FANCM function.

Tid1 (DNAJA3), a DnaJ co-chaperone, may promote degradation of oncogenic kinases. Tid1 has two isoforms, Tid1-L and Tid1-S, that may function differently. In this study, we investigated the role of the Tid1 isoforms in regulating EGFR signaling and lung cancer progression. We found that both Tid1-L and Tid1-S expressions were reduced in patients with non-small cell lung cancer compared with normal counterparts. Tid1-L expression correlated inversely with EGFR expression. Low Tid1-L/high EGFR expression predicted poor overall survival in lung adenocarcinoma patients. Tid1-L overexpression in lung cancer cells attenuated EGFR signaling and inhibited cell proliferation, colony formation and tumor growth in subcutaneous and orthotropic xenograft models. Conversely, depletion of Tid1 restored EGFR signaling and increased cell proliferation and colony formation. Tid1-L, but not Tid1-S interacted with EGFR/HSP70/HSP90 through the DnaJ domain, counteracting the EGFR regulatory function of HSP90 by causing EGFR ubiquitinylation and proteasomal degradation. Tid1-L inhibited EGFR signaling even more than the HSP90 inhibitor 17-allylamino-demethoxy geldanamycin. We concluded that Tid1-L acted as a tumor suppressor by inhibiting EGFR signaling through interaction with EGFR/HSP70/HSP90 and enhancing EGFR ubiquitinylation and degradation.

The MET oncogene, a crucial regulator of the genetic program known as "invasive growth" or "epithelial-mesenchymal transition," has recently emerged as a functional marker of glioblastoma stem cells. Here, we review findings that associate MET expression and activity with a specific, genetically defined glioblastoma stem cell subtype, and data showing how MET sustains the stem cell phenotype in glioblastoma and other tumors. Finally, we discuss issues related to identification of tumorigenic clones driven by MET in the context of genetically heterogeneous tumors and strategies aimed at eradicating cancer stem cells. Cancer Res; 73(11); 1-7. ©2013 AACR.

For tumor radiotherapy, the in vivo detection of early cellular responses is important for predicting therapeutic efficacy. Mn(2+) is used as a positive contrast agent in manganese-enhanced MRI (MEMRI) and is expected to behave as a mimic of Ca(2+) in many biologic systems. We conducted in vitro and in vivo MRI experiments with Mn(2+) to investigate whether MEMRI can be used to detect cell alterations as an early-phase tumor response after radiotherapy. Colon-26 cells or a subcutaneously grafted colon-26 tumor model were irradiated with 20 Gy of X-rays. One day after irradiation, a significant augmentation of G2-M-phase cells, indicating a cell-cycle arrest, was observed in the irradiated cells in comparison with the control cells, although both early and late apoptotic alterations were rarely observed. The MEMRI signal in radiation-exposed tumor cells (R1: 0.77 ± 0.01 s(-1)) was significantly lower than that in control cells (R1: 0.82 ± 0.01 s(-1)) in vitro. MEMRI signal reduction was also observed in the in vivo tumor model 24 hours after irradiation (R1 of radiation: 0.97 ± 0.02 s(-1), control: 1.10 ± 0.02 s(-1)), along with cell-cycle and proliferation alterations identified with immunostaining (cyclin D1 and Ki-67). Therefore, MEMRI after tumor radiotherapy was successfully used to detect cell alterations as an early-phase cellular response in vitro and in vivo. Cancer Res; 73(11); 1-9. ©2013 AACR.

Cell-cell communication, either in direct proximity or at a distance, generally occurs by receptor-ligand engagement and subsequent activation of downstream intracellular signaling cascades. This conventional, largely protein-based, model has long been considered necessary and sufficient to explain coordinate tissue and organismal function. Intriguing recent work indicates that many cells can also transfer RNA directly via cell-cell trafficking of nanometer-sized, lipid-bilayer vesicles. The distinct biogenesis pathways that give rise to the different vesicle types described to date are just beginning to be elucidated. Notwithstanding their diverse origin, all types of vesicles seem to contain a broad, cell-specific, nonrandom representation of cellular protein and RNA species. The cell-cell trafficking of coding and small noncoding RNAs in particular constitutes a new paradigm for the direct phenotypic modulation of cells in the local microenvironment and in distal organs. Here, we review the current understanding of RNA vesicle trafficking and its emerging role in cell-cell signaling. Cancer Res; 73(11); 1-6. ©2013 AACR.

Gleason score 7 (GS7) prostate cancer [tumors with both Gleason patterns 3 (GP3) and 4 (GP4)] portends a significantly more aggressive tumor than Gleason score 6 (GS6). It is, therefore, critical to understand the molecular relationship of adjacent GP3 and GP4 tumor cell populations and relate molecular abnormalities to disease progression. To decipher molecular relatedness, we used laser capture microdissection (LCM) and whole-genome amplification (WGA) to separately collect and amplify DNA from adjacent GP3 and GP4 cell populations from 14 cases of GS7 prostate cancer. We then carried out massively parallel mate-pair next generation sequencing (NGS) to examine the landscape of large chromosomal alterations. We identified four to 115 DNA breakpoints in GP3 and 17 to 480 in GP4. Our findings indicate that while GP3 and GP4 from the same tumor each possess unique breakpoints, they also share identical ones, indicating a common origin. Approximately 300 chromosomal breakpoints were localized to the regions affected in at least two tumors, whereas more than 3,000 were unique within the set of 14 tumors. TMPRSS2-ERG was the most recurrent rearrangement present in eight cases, in both GP3 and GP4. PTEN rearrangements were found in five of eight TMPRSS2-ERG fusion-positive cases in both GP3 and GP4. Hierarchical clustering analysis revealed that GP3 has greater breakpoint similarity to its partner GP4 compared with GP3 from different patients. We show evidence that LCM, WGA, and NGS of adjacent tumor regions provide an important tool in deciphering lineage relationships and discovering chromosomal alterations associated with tumor progression. Cancer Res; 73(11); 1-10. ©2013 AACR.

Vascular endothelial growth factor-C (VEGF-C) and VEGF-D were identified as lymphangiogenic growth factors and later shown to promote tumor metastasis, but their effects on carcinogenesis are poorly known. We have here studied the effects of VEGF-C and VEGF-D on tumor development in the murine multi-step chemical carcinogenesis model of squamous cell carcinoma (SCC) by using a soluble VEGF-C/D inhibitor. After topical treatment with a tumor initiator and repeated tumor promoter applications, transgenic mice expressing a soluble VEGF-C/D receptor (sVEGFR-3) in the skin developed significantly fewer squamous cell tumors with a delayed onset when compared to wild-type mice or mice expressing sVEGFR-3 lacking the ligand-binding site. Epidermal proliferation was reduced in the carcinogen treated transgenic skin, whereas epidermal keratinocyte proliferation in vitro was not affected by VEGF-C or VEGF-D, indicating indirect effects of sVEGFR-3 expression. Importantly, transgenic mouse skin was less sensitive to tumor promoter induced inflammation, with reduced angiogenesis and blood vessel leakage. Cutaneous leukocytes, especially macrophages, were reduced in transgenic skin without major changes in macrophage polarization or blood monocyte numbers. Several macrophage-associated cytokines were also reduced in transgenic papillomas, although the dermal macrophages themselves did not express VEGFR-3. These findings indicate that VEGF-C/D are involved in shaping the inflammatory tumor microenvironment that regulates early tumor progression. Our results support the use of VEGF-C/D blocking agents not only to inhibit metastatic progression, but also during the early stages of tumor growth.

Cisplatin is a widely used anticancer drug that acts by binding DNA and causing the formation of intrastrand and interstrand (ICL) cross-links, but the precise downstream effects of the latter damage are not well understood. In this study, we investigated the influence of cisplatin ICLs on synthetic nucleosomes that were platinated in a site-specific manner in vitro and on gene transcription in live mammalian cells. Nucleosome core particles (NCPs) that we constructed contained site-specific cisplatin 5'-d(G*pC)/5'-d(G*pC) ICLs, where the asterisk denotes the platinated nucleoside, to examine the influence of platinum lesions on the dynamic behavior of nucleosomes in solution. A cisplatin ICL, but not a 1,2-d(GpG) cross-link, significantly inhibited ATP-independent histone octamer-DNA sliding. We also used a novel linearization recircularization strategy described here to synthesize mammalian expression vectors containing site-specific cisplatin ICLs. Plasmid vectors were tested in live mammalian cells to study the transcription inhibition effects of cisplatin ICLs in the context of two different repair backgrounds. Cisplatin ICLs inhibit transcription as effectively as 1,2-d(GpG) cross-links. We determined that nucleotide excision repair plays a key role in the removal of cisplatin ICLs, acting in a replication-independent fashion. We also found that loss of mismatch repair function dramatically attenuates the transcription inhibition effects by cisplatin ICLs but not 1,2-d(GpG) intrastrand cross-links. Our results revealed the unique properties of cisplatin ICLs on nucleosome mobility and on transcription, and they defined how these adducts act in a manner completely different from that used for cisplatin 1,2-d(GpG) cross-links. These new findings provide direct support for a role of ICLs in the pharmacological activities of cisplatin, despite the lower frequency of their formation.

ShcA (SHC1) is an adapter protein that possesses an SH2 and a PTB phosphotyrosine binding motif. ShcA generally utilizes its PTB domain to engage activated receptor tyrosine kinases (RTKs), but there has not been a definitive determination of the role of this domain in tumorigenesis. To address this question, we employed a ShcA mutant (R175Q) that no longer binds phospho-tyrosine residues via its PTB domain. Here we report that transgenic expression of this mutant delays mammary tumor onset in the MMTV-PyMT mouse model of breast cancer. Paradoxically, we observed a robust increase in the growth and angiogenesis of mammary tumors expressing ShcR175Q, which displayed increased secretion of fibronectin and expression of integrin α5/β1, the principle fibronectin receptor. Sustained integrin engagement activated Src, which in turn phosphorylated pro-angiogenic RTKs, including PDGFR, FGFR and Met, leading to increased VEGF secretion from ShcR175Q-expressing breast cancer cells. We defined a ShcR175Q-dependent gene signature that could stratify breast cancer patients with a high microvessel density. This study offers the first in vivo evidence of a critical role for intracellular signaling pathways downstream of the ShcA PTB domain, which both positively and negatively regulate tumorigenesis during various stages of breast cancer progression.