Baseline MAPK signaling activity confers intrinsic radioresistance to KRAS-mutant colorectal carcinoma cells by rapid upregulation of heterogeneous nuclear ribonucleoprotein K (hnRNP K).

Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is overexpressed in malignant tumors and involved in DNA damage response upon ionizing radiation (IR). Here, we investigate its role in radioresistance of colorectal carcinoma (CRC) and evaluate a pharmacological approach to enhance CRC radiosensitivity via downregulation of hnRNP K. We show that hnRNP K is overexpressed in CRC tissue specimens and upregulated in response to IR in vitro, which occurs faster in KRAS-mutant CRC cells. HnRNP K knockdown impairs cell survival, cell cycle progression and KRAS-dependent radioresistance and increases apoptosis. Using the chicken chorioallantoic membrane assay, a decrease in xenograft tumor growth and radioresistance upon hnRNP K depletion could be verified in vivo, and comparable effects were achieved by suppression of hnRNP K expression using the MEK inhibitor MEK162 (Binimetinib). In summary, KRAS-mutant CRC shows intrinsic radioresistance along with rapid upregulation of hnRNP K in response to IR that can effectively be targeted by MEK inhibition. Our results point towards a possible use of MAPK pathway inhibitors to decrease radioresistance of KRAS-mutant CRC via downregulation of hnRNP K.

Cyr61 and YB-1 are novel interacting partners of uPAR and elevate the malignancy of triple-negative breast cancer.

The triple-negative breast cancer (TNBC) is a very aggressive tumor type often occurring in young women and is associated with a bad prognosis for the patients. TNBC lacks established targets for breast cancer therapy, such as the estrogen receptor (ER), progesterone receptor (PR) and the human epidermal growth factor receptor 2 (HER2). Therefore, novel therapeutic targets and strategies are needed for an improved treatment of this breast cancer subtype. TNBC and respective cell lines often overexpress proteins of the urokinase plasminogen activator system (uPAS) including uPA, its receptor uPAR and inhibitor PAI-1, which together with co-factors contribute to the malignancy of TNBC. Here, two novel interacting partners of uPAR, the cysteine-rich angiogenic inducer 61 (Cyr61) and the Y-box-binding protein 1 (YB-1) were identified and their differential expression demonstrated in TNBC cells as well as in tumors. In the TNBC cohort, both interactors significantly correlated with expression levels of cathepsin B, c-Met and the tumor grade. In addition, expression levels of Cyr61 significantly correlated with cathepsin D (p=0.03), insulin receptor (p≤0.001), insulin-like growth factor receptor 1 (IGF1R, p=0.015) and also with YB-1 (p=0.0004) levels. The interactions of uPAR with Cyr61 significantly correlated with expression levels of tumor-promoting biomarkers including plasminogen (p=0.0014), cathepsin B (p=0.032), c-Met (p=0.0192) as well as with the tumor grade (p=0.02). In multivariate survival analysis, YB-1 showed independent prognostic value (p=0.01). As the novel interacting partners, also together with uPAR, contribute to tumor progression and metastasis, both may be potential therapeutic targets in breast cancer.

Proteomic Profiling Suggests Central Role Of STAT Signaling during Retinal Degeneration in the rd10 Mouse Model.

The rd10 mouse is a model of retinitis pigmentosa characterized by the dysfunction of a rod-photoreceptor-specific phosphodiesterase. Compared to the rd1 mouse, retinal degeneration in the rd10 mouse begins later in age with a milder phenotype, making it ideal for investigating cell death and neuroprotective mechanisms. Alterations in the rd10 retina proteome at pre-, peak-, and postdegenerative time points were examined using a modified high-recovery filter-aided sample preparation (FASP) method in combination with label-free quantitative mass spectrometry, generating a proteomic data set on almost 3000 proteins. Our data confirmed a period of protein expression similar to age-matched wild-type mice predegeneration, with decreases in proteins associated with phototransduction and increases in signaling proteins at peak- and postdegenerative stages. A total of 57 proteins were differentially expressed in the rd10 retinae during peak-degeneration, compared to those in wild-type mice after stringent FDR correction (q < 0.05). Network analysis separated these proteins into one cluster of down-regulated photoreceptor proteins and one of up-regulated signaling proteins centered around GFAP, STAT3, and STAT1. This is the first study to identify alterations in STAT1 in the rd10 mouse, which were confirmed with gene expression and immunoblotting experiments, underpinning the efficacy of our approach. This unique proteomic data set on protein dynamics during retinal degeneration could serve as an information source for vision research in the future.

Radiosensitization and downregulation of heterogeneous nuclear ribonucleoprotein K (hnRNP K) upon inhibition of mitogen/extracellular signal-regulated kinase (MEK) in malignant melanoma cells.

BACKGROUND: Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is an important cofactor in the p53-mediated DNA damage response pathway upon ionizing radiation (IR) and exerts anti-apoptotic effects also independent of p53 pathway activation. Furthermore, hnRNP K is overexpressed in various neoplasms including malignant melanoma (MM). Here, we investigate the role of hnRNP K in the radioresistance of MM cells. METHODS AND RESULTS: Our results show cytoplasmic expression of hnRNP K in human MM surgical specimens, but not in benign nevi, and a quick dose- and time-dependent upregulation in response to IR accompanied by cytoplasmic redistribution of the protein in the IPC-298 cellular tumor model carrying an activating NRAS mutation (p.Q61L). SiRNA-based knockdown of hnRNP K induced a delayed decline in γH2AX/53BP1-positive DNA repair foci upon IR. Pharmacological interference with MAPK signaling abrogated ERK phosphorylation, diminished cellular hnRNP K levels, impaired γH2AX/53BP1-foci repair and proliferative capability and increased apoptosis comparable to the observed hnRNP K knockdown phenotype in IPC-298 cells. CONCLUSIONS: Our results indicate that pharmacological interference with MAPK signaling increases vulnerability of NRAS-mutant malignant melanoma cells to ionizing radiation along with downregulation of endogenous hnRNP K and point towards a possible use for combined MEK inhibition and localized radiation therapy of MM in the NRAS-mutant setting where BRAF inhibitors offer no clinical benefit.

Proper cerebellar development requires expression of ß1-integrin in Bergmann glia, but not in granule neurons.

ß1-class integrins play essential roles both in developmental biology as well as in cancer. Particularly, a Nestin-driven deletion of ß1-integrin receptors results in severe abnormalities of brain development including a laminar disorganization of cerebellar granule neurons. However, since Nestin is expressed in all kinds of neural precursors, these data do not allow conclusions to be drawn about the role of ß1-integrins in distinct neuronal and glial cell types. By generating conditional knockout mice using granule cell-specific Math1-promoter sequences, we show here that the expression of ß1-integrins in granule neurons is dispensable for the development of the cerebellum. Also, deletion of ß1-integrin from tumors that arise in a mouse model of granule cell precursor-derived medulloblastoma did not result in a significant survival benefit. Last, expression levels of ß1-integrin in human medulloblastoma samples did not predict patient's outcome. However, a ß1-integrin knockout using hGFAP-promoter sequences led to cerebellar hypoplasia, inappropriate positioning of Bergmann glia cells in the molecular layer, undirected outgrowth of radial glia fibers, and granule cell ectopia. We therefore conclude that ß1-integrin expression in cerebellar granule neurons is not essential during normal development or medulloblastoma formation. In fact, it is the expression of ß1-integrin in glia that is crucial for the proper development of the cerebellar cortex.

Expression of FoxM1 is required for the proliferation of medulloblastoma cells and indicates worse survival of patients.

PURPOSE: The transcription factor Forkhead box M1 (FoxM1) is a key regulator of cell-cycle progression. It is involved in the development of multiple organs, and we have previously reported on its important role for the mitotic entry of cerebellar granule neuron precursors. Constitutive expression of FoxM1 is required for the growth of multiple cancer types. This study aimed to determine its role in medulloblastoma, the most frequent malignant brain tumor in childhood that can derive from cerebellar granule neuron precursors. EXPERIMENTAL DESIGN: We evaluated the expression of FoxM1 together with its prognostic value in two independent series of human medulloblastoma samples using immunohistochemistry (n = 43) and gene expression arrays (n = 193). The functional impact of FoxM1 expression was characterized by knockdown experiments in four human medulloblastoma cell lines, and the thiazole antibiotic siomycin A was tested to downregulate FoxM1 and inhibit tumor cell growth. RESULTS: FoxM1 was highly expressed in all subtypes of medulloblastoma. Importantly, expression levels of FoxM1 significantly correlated with unfavorable clinical outcome in univariate analysis (P = 0.0005), and FoxM1 was identified as an independent prognostic marker by multivariate analysis (P = 0.037). Knockdown of FoxM1 in medulloblastoma cell lines resulted in a significant decrease of cell viability which was caused by a failure in mitotic spindle formation and caspase-dependent mitotic catastrophe. Siomycin A significantly inhibited the expression of FoxM1 and the growth of medulloblastoma cells. CONCLUSIONS: FoxM1 may be used as an additional prognostic marker and may represent a potential novel target to treat patients suffering from medulloblastoma.

Dexamethasone destabilizes Nmyc to inhibit the growth of hedgehog-associated medulloblastoma.

Mouse studies indicate that the synthetic glucocorticoid dexamethasone (Dex) impairs the proliferation of granule neuron precursors in the cerebellum, which are transformed to medulloblastoma by activation of Sonic hedgehog (Shh) signaling. Here, we show that Dex treatment also inhibits Shh-induced tumor growth, enhancing the survival of tumor-prone transgenic mice. We found that Nmyc was specifically required in granule cells for Shh-induced tumorigenesis and that Dex acted to reduce Nmyc protein levels. Moreover, we found that Dex-induced destabilization of Nmyc is mediated by activation of glycogen synthase kinase 3beta, which targets Nmyc for proteasomal degradation. Together, our findings show that Dex antagonizes Shh signaling downstream of Smoothened in medulloblastoma.