Comparative analysis of cancer cell responses to targeted radionuclide therapy (TRT) and external beam radiotherapy (EBRT)

The vast majority of our knowledge regarding cancer radiobiology and the activation of radioresistance mechanisms emerged from studies using external beam radiation therapy (EBRT). Yet, less is known about the cancer response to internal targeted radionuclide therapy (TRT). Our comparative phosphoproteomics analyzed cellular responses to TRT with lutetium-177-labeled minigastrin analogue [$^{177}$Lu]Lu-PP-F11N (β-emitter) and EBRT (ɣ-rays) in CCKBR-positive cancer cells. Activation of DNA damage response by p53 was induced by both types of radiotherapy, whereas TRT robustly increased activation of signaling pathways including epidermal growth factor receptor (EGFR), mitogen-activated protein kinases (MAPKs) or integrin receptor. Inhibition of EGFR or integrin signaling sensitized cancer cells to radiolabeled minigastrin. In vivo, EGFR inhibitor erlotinib increased therapeutic response to [$^{177}$Lu]Lu-PP-F11N and median survival of A431/CCKBR-tumor bearing nude mice. In summary, our study explores a complex scenario of cancer responses to different types of irradiation and pinpoints the radiosensitizing strategy, based on the targeting survival pathways, which are activated by TRT

Lack of NWC protein (c11orf74 homolog) in murine spermatogenesis results in reduced sperm competitiveness and impaired ability to fertilize egg cells in vitro

<div><p>NWC is an uncharacterised protein containing three strongly conserved domains not found in any other known protein. Previously, we reported that the NWC protein is detected in cells in the germinal layer in murine testes (strain: C57BL/6), and its knockout results in no obvious phenotype. We determined the NWC expression pattern during spermatogenesis, and found this protein in spermatocytes and round spermatids, but not in epididymal sperm. Although NWC knockout males are fertile, we further characterised their reproductive potential employing non-standard mating that better simulates the natural conditions by including sperm competition. Such an approach revealed that the sperm of knockout males fail to successfully compete with control sperm. After analysing selected characteristics of the male reproductive system, we found that <i>NWC</i> knockout sperm had a reduced ability to fertilize cumulus-intact eggs during IVF. This is the first report describing a subtle phenotype of <i>NWC</i> knockout mice that could be detected under non-standard mating conditions. Our results indicate that NWC plays an important role in spermatogenesis and its deficiency results in the production of functionally impaired sperm.</p></div

The INT6 Cancer Gene and MEK Signaling Pathways Converge during Zebrafish Development

BACKGROUND: Int-6 (integration site 6) was identified as an oncogene in a screen of tumorigenic mouse mammary tumor virus (MMTV) insertions. INT6 expression is altered in human cancers, but the precise role of disrupted INT6 in tumorigenesis remains unclear, and an animal model to study Int-6 physiological function has been lacking. PRINCIPAL FINDINGS: Here, we create an in vivo model of Int6 function in zebrafish, and through genetic and chemical-genetic approaches implicate Int6 as a tissue-specific modulator of MEK-ERK signaling. We find that Int6 is required for normal expression of MEK1 protein in human cells, and for Erk signaling in zebrafish embryos. Loss of either Int6 or Mek signaling causes defects in craniofacial development, and Int6 and Erk-signaling have overlapping domains of tissue expression. SIGNIFICANCE: Our results provide new insight into the physiological role of vertebrate Int6, and have implications for the treatment of human tumors displaying altered INT6 expression

Analyse differentiell exprimierter Gene im humanen Prostatakarzinom

Prostatakrebs ist der am häufigsten prognostizierte solide Tumor und die zweithäufigste Ursache für Krebstod bei Männern in den westlichen Ländern. Eine der Schlüsselaufgaben der Prostatakrebsforschung ist es, molekulare Marker zu entwickeln, die effektiv Progression und Malignität von Prostatatumoren detektieren und unterscheiden, sowie Einsichten in die Entwicklung und das Verhalten von Prostatatumoren ermöglichen können. Aus der Literatur ist bekannt, dass der Insulin-like growth factor I receptor (IGF-IR) eine wichtige Rolle in der zellulären Homöostase von Prostatakarzinomen spielt. Daher wurde die antisense-RNA-Strategie genutzt, um die endogene IGF-IR Genexpression in menschlichen Prostatakarzinom PC-3 Zellen zu reduzieren, was in einer signifikanten Suppression der Zellinvasion und Proliferation sowie in einer Erhöhung der Zellapoptose von PC-3 Zellen resultierte. Weiterhin wurde gezeigt, dass eine direkte Korrelation zwischen der Inhibierung der IGF-IR Genexpression und der Hochregulierung von IGFBP-3 oder der Herunterregulierung der Expression von MMP-2 in androgen-unabhängigen PC-3 Zellen existiert. Zusätzlich wurden die Expressionen sowohl von IGF-IR als auch von IGFBP-3 mittels quantitativer real time RT-PCR an RNA aus lasermikrodissektierten zusammengehörigen normalen und Tumor-Prostataepithelgeweben von 12 Patienten analysiert. Diese Untersuchungen zeigten, dass IGF-IR in den meisten Prostatakarzinomen (9 von 12) hochreguliert und dass IGFBP-3 in allen der Prostatakarzinome herunterreguliert ist. Weiterhin wurde die Hochregulierung der Genexpression von IGFBP-3 mittels eines cancer profiling arrays in folgenden Karzinomen bestimmt: Nieren-, Lungen-, Mastdarm-, Dickdarm-, Magen und Schilddrüsenkrebs. Im Gegensatz dazu wurde eine Herunterregulierung in Brust-, Uterus- und Ovarialkrebs festgestellt. Diese Ergebnisse deuten auf eine wichtige Rolle von IGF-IR und IGFBP-3 in der zellulären Homöstase von Prostatakarzinomen hin und liefern eine weitere Grundlage, den IGF-IR als Zielmolekül für eine potentielle Behandlung von Prostatakrebs einzusetzen, sowie die IGF-IR-aktivierten Signalwege besser zu verstehen. Als nächstes wurden die Expressionslevel von über 400 krebs-verwandten Genen in einer Reihe von Prostatatumoren und zugehörigen normalen Prostatageweben mittels cDNA-Array-Technik verglichen, um die differentielle Genexpression von putativen Markern für Prostatatumoren zu analysieren. Insgesamt wurden 46 differentiell exprimierte Gene identifiziert, die entweder hoch- oder herunterreguliert im Prostatakarzinom sind. Von diesen differentiell exprimierten Genen wurde von 7 Genen (Bax-Inhibitor-1, Komplementkomponente C1s, Ferritin heavy chain, MAT8 Protein, Peptidyl-Prolyl cis-trans Isomerase A, RNA-binding protein regulatory subunit DJ-1 protein und V-ATP Synthase Untereinheit F), die eine hochregulierte Expression in menschlichen Prostatakarzinomen zeigten, die Überexpression in Prostatakrebs mittels real time RT-PCR an Gesamttumor-RNA bestätigt. Die Überexpression von Bax-Inhibitor-1 (BI-1) in Prostatakarzinomen wurde mittels Northern Blot-Analysen an Gesamttumor-RNA und mittels real time RT-PCR an RNA von 13 lasermikrodissektierten Tumorgewebeproben der Prostata bestätigt. Zusätzlich wurde die hochregulierte Expression vom BI-1 Gen in folgenden Karzinomen bestimmt: Ovarial-, Uterus-, Prostata- und Brustkrebs, hingegen wurde eine herunterregulierte Expression in Nieren-, Lungen-, Dickdarm-, Magen- und Mastdarmkrebs festgestellt. Um weiterhin die Funktion von BI-1 in vitro zu untersuchen, wurden menschliche androgen-unabhängige PC-3 und androgen-abhängige LNCaP Prostatakarzinomzellen mit small interfering RNA (siRNA) Oligonukleotiden gegen das BI-1 Gen transfiziert, was zu einer spezifischen Herunterregulierung der BI-1-Expression führte. Ferner verursachte die Transfektion von PC-3 und LNCaP-Zellen mit BI-1-sequenzspezifischen siRNAs eine signifikante Erhöhung von zellulärer Apoptose sowie Nekrose. Zusammengefasst deuten die derzeitigen Ergebnisse darauf hin, dass das menschliche BI-1 Gen das Potential besitzt, als Expressionsmarker von Prostatakrebs zu fungieren und ein neues Ziel für die Entwicklung von therapeutischen Strategien gegen Prostatakrebs darstellt

Therapeutic Response of CCKBR-Positive Tumors to Combinatory Treatment with Everolimus and the Radiolabeled Minigastrin Analogue [177Lu]Lu-PP-F11N

The inhibition of the mammalian target of rapamycin complex 1 (mTORC1) by everolimus (RAD001) was recently shown to enhance the tumor uptake of radiolabeled minigastrin. In this paper, we investigate if this finding can improve the in vivo therapeutic response to [177Lu]Lu-PP-F11N treatment. The N-terminal DOTA-conjugated gastrin analogue PP-F11N (DOTA-(DGlu)6-Ala-Tyr-Gly-Trp-Nle-Asp-Phe) was used to evaluate treatment efficacy in the human A431/CCKBR xenograft nude mouse model in combination with RAD001. Both RAD001 and [177Lu]Lu-PP-F11N single treatments as well as their combination inhibited tumor growth and increased survival. In concomitantly treated mice, the average tumor size and median survival time were significantly reduced and extended, respectively, as compared to the monotherapies. The histological analysis of kidney and stomach dissected after treatment with RAD001 and [177Lu]Lu-PP-F11N did not indicate significant adverse effects. In conclusion, our study data demonstrate the potential of mTORC1 inhibition to substantially improve the therapeutic efficacy of radiolabeled minigastrin analogues in CCKBR-positive cancers

Targeting mTORC1 Activity to Improve Efficacy of Radioligand Therapy in Cancer

Radioligand therapy (RLT) represents an effective strategy to treat malignancy by cancer-selective delivery of radioactivity following systemic application. Despite recent therapeutic successes, cancer radioresistance and insufficient delivery of the radioactive ligands, as well as cytotoxicity to healthy organs, significantly impairs clinical efficacy. To improve disease management while minimizing toxicity, in recent years, the combination of RLT with molecular targeted therapies against cancer signaling networks showed encouraging outcomes. Characterization of the key deregulated oncogenic signaling pathways revealed their convergence to activate the mammalian target of rapamycin (mTOR), in which signaling plays an essential role in the regulation of cancer growth and survival. Therapeutic interference with hyperactivated mTOR pathways was extensively studied and led to the development of mTOR inhibitors for clinical applications. In this review, we outline the regulation and oncogenic role of mTOR signaling, as well as recapitulate and discuss mTOR complex 1 (mTORC1) inhibition to improve the efficacy of RLT in cancer.ISSN:2072-669

Therapeutic Response of CCKBR-Positive Tumors to Combinatory Treatment with Everolimus and the Radiolabeled Minigastrin Analogue [177Lu]Lu-PP-F11

The inhibition of the mammalian target of rapamycin complex 1 (mTORC1) by everolimus (RAD001) was recently shown to enhance the tumor uptake of radiolabeled minigastrin. In this paper, we investigate if this finding can improve the in vivo therapeutic response to [177Lu]Lu-PP-F11N treatment. The N-terminal DOTA-conjugated gastrin analogue PP-F11N (DOTA-(DGlu)6-Ala-Tyr-Gly-Trp-Nle-Asp-Phe) was used to evaluate treatment efficacy in the human A431/CCKBR xenograft nude mouse model in combination with RAD001. Both RAD001 and [177Lu]Lu-PP-F11N single treatments as well as their combination inhibited tumor growth and increased survival. In concomitantly treated mice, the average tumor size and median survival time were significantly reduced and extended, respectively, as compared to the monotherapies. The histological analysis of kidney and stomach dissected after treatment with RAD001 and [177Lu]Lu-PP-F11N did not indicate significant adverse effects. In conclusion, our study data demonstrate the potential of mTORC1 inhibition to substantially improve the therapeutic efficacy of radiolabeled minigastrin analogues in CCKBR-positive cancers

Evaluation of Actinium-225 Labeled Minigastrin Analogue [225Ac]Ac-DOTA-PP-F11N for Targeted Alpha Particle Therapy

The overexpression of cholecystokinin B receptor (CCKBR) in human cancers led to the development of radiolabeled minigastrin analogues for targeted radionuclide therapy, which aims to deliver cytotoxic radiation specifically to cancer cells. Alpha emitters (e.g., actinium-225) possess high potency in cancer cell-killing and hold promise for the treatment of malignant tumors. In these preclinical studies, we developed and evaluated CCKBR-targeted alpha particle therapy. The cellular uptake and cytotoxic effect of actinium-225 labeled and HPLC-purified minigastrin analogue [225Ac]Ac-PP-F11N were characterized in the human squamous cancer A431 cells transfected with CCKBR. Nude mice bearing A431/CCKBR tumors were used for biodistribution and therapy studies followed by histological analysis and SPECT/CT imaging. In vitro, [225Ac]Ac-PP-F11N showed CCKBR-specific and efficient internalization rate and potent cytotoxicity. The biodistribution studies of [225Ac]Ac-PP-F11N revealed CCKBR-specific uptake in tumors, whereas the therapeutic studies demonstrated dose-dependent inhibition of tumor growth and extended mean survival time, without apparent toxicity. The histological analysis of kidney and stomach indicated no severe adverse effects after [225Ac]Ac-PP-F11N administration. The post-therapy SPECT-CT images with [111In]In-PP-F11N confirmed no CCKBR-positive tumor left in the mice with complete remission. In conclusion, our study demonstrates therapeutic efficacy of [225Ac]Ac-PP-F11N without acute radiotoxicity in CCKBR-positive cancer model

MAP kinase-interacting kinase 1 regulates SMAD2-dependent TGF-β signaling pathway in human glioblastoma

Glioblastoma multiforme (GBM) is the most common aggressive brain cancer with a median survival of approximately 1 year. In a search for novel molecular targets that could be therapeutically developed, our kinome-focused microarray analysis identified the MAP (mitogen-activated protein) kinase-interacting kinase 1 (MNK1) as an attractive theranostic candidate. MNK1 overexpression was confirmed in both primary GBMs and glioma cell lines. Inhibition of MNK1 activity in GBM cells by the small molecule CGP57380 suppressed eIF4E phosphorylation, proliferation, and colony formation whereas concomitant treatment with CGP57380 and the mTOR inhibitor rapamycin accentuated growth inhibition and cell-cycle arrest. siRNA-mediated knockdown of MNK1 expression reduced proliferation of cells incubated with rapamycin. Conversely, overexpression of full-length MNK1 reduced rapamycin-induced growth inhibition. Analysis of polysomal profiles revealed inhibition of translation in CGP57380 and rapamycin-treated cells. Microarray analysis of total and polysomal RNA from MNK1-depleted GBM cells identified mRNAs involved in regulation of TGF-? pathway. Translation of SMAD2 mRNA as well as TGF-?-induced cell motility and vimentin expression was regulated by MNK1 signaling. Tissue microarray analysis revealed a positive correlation between the immunohistochemical staining of MNK1 and SMAD2. Taken together, our findings offer insights into how MNK1 pathways control translation of cancer-related mRNAs including SMAD2, a key component of the TGF-? signaling pathway. Furthermore, they suggest MNK1-controlled translational pathways in targeted strategies to more effectively treat GBM