A quantitative method for detection of circulating fms related tyrosine kinase 3 (FLT-3) in acute myeloid leukemia (AML) patients.

FMS related tyrosine kinase 3 (FLT-3) is a tyrosine kinase expressed in early hematopoietic precursor cells and has roles in survival, proliferation, and differentiation. Bone marrow expression and mutagenic analysis of FLT-3 in Acute Myeloid Leukemia (AML) patients is well-characterized. However, the levels of circulating FLT-3 in serum have not been previously described. In this study we describe a quantitative electrochemiluminescent immunoassay that detects FLT-3 in human serum. Using this method we find that AML patients have elevated levels of circulating FLT-3 and these levels correlated to the percent blast counts in the bone marrow (BM).

Erratum: Induction of LIFR confers a dormancy phenotype in breast cancer cells disseminated to the bone marrow.

This corrects the article DOI: 10.1038/ncb3408.

Induction of LIFR confers a dormancy phenotype in breast cancer cells disseminated to the bone marrow.

Breast cancer cells frequently home to the bone marrow, where they may enter a dormant state before forming a bone metastasis. Several members of the interleukin-6 (IL-6) cytokine family are implicated in breast cancer bone colonization, but the role for the IL-6 cytokine leukaemia inhibitory factor (LIF) in this process is unknown. We tested the hypothesis that LIF provides a pro-dormancy signal to breast cancer cells in the bone. In breast cancer patients, LIF receptor (LIFR) levels are lower with bone metastases and are significantly and inversely correlated with patient outcome and hypoxia gene activity. Hypoxia also reduces the LIFR:STAT3:SOCS3 signalling pathway in breast cancer cells. Loss of the LIFR or STAT3 enables otherwise dormant breast cancer cells to downregulate dormancy-, quiescence- and cancer stem cell-associated genes, and to proliferate in and specifically colonize the bone, suggesting that LIFR:STAT3 signalling confers a dormancy phenotype in breast cancer cells disseminated to bone.

Hypoxic induction of AKAP12 variant 2 shifts PKA-mediated protein phosphorylation to enhance migration and metastasis of melanoma cells.

Scaffold proteins are critical hubs within cells that have the ability to modulate upstream signaling molecules and their downstream effectors to fine-tune biological responses. Although they can serve as focal points for association of signaling molecules and downstream pathways that regulate tumorigenesis, little is known about how the tumor microenvironment affects the expression and activity of scaffold proteins. This study demonstrates that hypoxia, a common element of solid tumors harboring low oxygen levels, regulates expression of a specific variant of the scaffold protein AKAP12 (A-kinase anchor protein 12), AKAP12v2, in metastatic melanoma. In turn, through a kinome-wide phosphoproteomic and MS study, we demonstrate that this scaffolding protein regulates a shift in protein kinase A (PKA)-mediated phosphorylation events under hypoxia, causing alterations in tumor cell invasion and migration in vitro, as well as metastasis in an in vivo orthotopic model of melanoma. Mechanistically, the shift in AKAP12-dependent PKA-mediated phosphorylations under hypoxia is due to changes in AKAP12 localization vs. structural differences between its two variants. Importantly, our work defines a mechanism through which a scaffold protein can be regulated by the tumor microenvironment and further explains how a tumor cell can coordinate many critical signaling pathways that are essential for tumor growth through one individual scaffolding protein.

Direct regulation of GAS6/AXL signaling by HIF promotes renal metastasis through SRC and MET.

Dysregulation of the von Hippel-Lindau/hypoxia-inducible transcription factor (HIF) signaling pathway promotes clear cell renal cell carcinoma (ccRCC) progression and metastasis. The protein kinase GAS6/AXL signaling pathway has recently been implicated as an essential mediator of metastasis and receptor tyrosine kinase crosstalk in cancer. Here we establish a molecular link between HIF stabilization and induction of AXL receptor expression in metastatic ccRCC. We found that HIF-1 and HIF-2 directly activate the expression of AXL by binding to the hypoxia-response element in the AXL proximal promoter. Importantly, genetic and therapeutic inactivation of AXL signaling in metastatic ccRCC cells reversed the invasive and metastatic phenotype in vivo. Furthermore, we define a pathway by which GAS6/AXL signaling uses lateral activation of the met proto-oncogene (MET) through SRC proto-oncogene nonreceptor tyrosine kinase to maximize cellular invasion. Clinically, AXL expression in primary tumors of ccRCC patients correlates with aggressive tumor behavior and patient lethality. These findings provide an alternative model for SRC and MET activation by growth arrest-specific 6 in ccRCC and identify AXL as a therapeutic target driving the aggressive phenotype in renal clear cell carcinoma.

TBC1D16 is a Rab4A GTPase activating protein that regulates receptor recycling and EGF receptor signaling.

Rab4A is a master regulator of receptor recycling from endocytic compartments to the plasma membrane. The protein TBC1D16 is up-regulated in melanoma, and TBC1D16-overexpressing melanoma cells are dependent on TBC1D16. We show here that TBC1D16 enhances the intrinsic rate of GTP hydrolysis by Rab4A. TBC1D16 is both cytosolic and membrane associated; the membrane-associated pool colocalizes with transferrin and EGF receptors (EGFRs) and early endosome antigen 1, but not with LAMP1 protein. Expression of two TBC1D16 isoforms, but not the inactive R494A mutant, reduces transferrin receptor recycling but has no effect on transferrin receptor internalization. Expression of TBC1D16 alters GFP-Rab4A membrane localization. In HeLa cells, overexpression of TBC1D16 enhances EGF-stimulated EGFR degradation, concomitant with decreased EGFR levels and signaling. Thus, TBC1D16 is a GTPase activating protein for Rab4A that regulates transferrin receptor recycling and EGFR trafficking and signaling.

VHL loss in renal cell carcinoma leads to up-regulation of CUB domain-containing protein 1 to stimulate PKC{delta}-driven migration.

A common genetic mutation found in clear cell renal cell carcinoma (CC-RCC) is the loss of the von Hippel-Lindau (VHL) gene, which results in stabilization of hypoxia-inducible factors (HIFs), and contributes to cancer progression and metastasis. CUB-domain-containing protein 1 (CDCP1) was shown to promote metastasis in scirrhous and lung adenocarcinomas as well as in prostate cancer. In this study, we established a molecular mechanism linking VHL loss to induction of the CDCP1 gene through the HIF-1/2 pathway in renal cancer. Also, we report that Fyn, which forms a complex with CDCP1 and mediates its signaling to PKCδ, is a HIF-1 target gene. Mechanistically, we found that CDCP1 specifically regulates phosphorylation of PKCδ, but not of focal adhesion kinase or Crk-associated substrate. Signal transduction from CDCP1 to PKCδ leads to its activation, increasing migration of CC-RCC. Furthermore, patient survival can be stratified by CDCP1 expression at the cell surface of the tumor. Taken together, our data indicates that CDCP1 protein might serve as a therapeutic target for CC-RCC.

Hypoxia, inflammation, and the tumor microenvironment in metastatic disease.

Metastasis, the leading cause of cancer deaths, is an intricate process involving many important tumor and stromal proteins that have yet to be fully defined. This review discusses critical components necessary for the metastatic cascade, including hypoxia, inflammation, and the tumor microenvironment. More specifically, this review focuses on tumor cell and stroma interactions, which allow cell detachment from a primary tumor, intravasation to the blood stream, and extravasation at a distant site where cells can seed and tumor metastases can form. Central players involved in this process and discussed in this review include integrins, matrix metalloproteinases, and soluble growth factors/matrix proteins, including the connective tissue growth factor and lysyl oxidase.

TbetaRIII suppresses non-small cell lung cancer invasiveness and tumorigenicity.

The transforming growth factor-beta (TGF-beta) superfamily has essential roles in lung development, regulating cell proliferation, branching morphogenesis, differentiation and apoptosis. Although most lung cancers become resistant to the tumor suppressor effects of TGF-beta, and loss or mutation of one of the components of the TGF-beta signaling pathway, including TbetaRII, Smad2 and Smad4 have been reported, mutations are not common in non-small cell lung cancer (NSCLC). Here we demonstrate that the TGF-beta superfamily co-receptor, the type III TGF-beta receptor (TbetaRIII or betaglycan) is lost in the majority of NSCLC specimens at the mRNA and protein levels, with loss correlating with increased tumor grade and disease progression. Loss of heterozygosity at the TGFBR3 genomic locus occurs in 38.5% of NSCLC specimens and correlates with decreased TbetaRIII expression, suggesting loss of heterozygosity as one mechanism for TbetaRIII loss. In the H460 cell model of NSCLC, restoring TbetaRIII expression decreased colony formation in soft agar. In the A549 cell model of NSCLC, restoring TbetaRIII expression significantly decreased cellular migration and invasion through Matrigel, in the presence and absence of TGF-beta1, and decreased tumorigenicity in vivo. In a reciprocal manner, shRNA-mediated silencing of endogenous TbetaRIII expression enhanced invasion through Matrigel. Mechanistically, TbetaRIII functions, at least in part, through undergoing ectodomain shedding, generating soluble TbetaRIII, which is able to inhibit cellular invasiveness. Taken together, these results support TbetaRIII as a novel tumor suppressor gene that is commonly lost in NSCLC resulting in a functional increase in cellular migration, invasion and anchorage-independent growth of lung cancer cells.

The type III transforming growth factor-beta receptor as a novel tumor suppressor gene in prostate cancer.

The transforming growth factor-beta (TGF-beta) signaling pathway has an important role in regulating normal prostate epithelium, inhibiting proliferation, differentiation, and both androgen deprivation-induced and androgen-independent apoptosis. During prostate cancer formation, most prostate cancer cells become resistant to these homeostatic effects of TGF-beta. Although the loss of expression of either the type I (TbetaRI) or type II (TbetaRII) TGF-beta receptor has been documented in approximately 30% of prostate cancers, most prostate cancers become TGF-beta resistant without mutation or deletion of TbetaRI, TbetaRII, or Smads2, 3, and 4, and thus, the mechanism of resistance remains to be defined. Here, we show that type III TGF-beta receptor (TbetaRIII or betaglycan) expression is decreased or lost in the majority of human prostate cancers as compared with benign prostate tissue at both the mRNA and protein level. Loss of TbetaRIII expression correlates with advancing tumor stage and a higher probability of prostate-specific antigen (PSA) recurrence, suggesting a role in prostate cancer progression. The loss of TbetaRIII expression is mediated by the loss of heterozygosity at the TGFBR3 genomic locus and epigenetic regulation of the TbetaRIII promoter. Functionally, restoring TbetaRIII expression in prostate cancer cells potently decreases cell motility and cell invasion through Matrigel in vitro and prostate tumorigenicity in vivo. Taken together, these studies define the loss of TbetaRIII expression as a common event in human prostate cancer and suggest that this loss is important for prostate cancer progression through effects on cell motility, invasiveness, and tumorigenicity.