Gender-specific changes of the gut microbiome correlate with tumor development in murine models of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a dismal outcome. To improve understanding of sequential microbiome changes during PDAC development we analyzed mouse models of pancreatic carcinogenesis (KC mice recapitulating pre-invasive PanIN formation, as well as KPC mice recapitulating invasive PDAC) during early tumor development and subsequent tumor progression. Diversity and community composition were analyzed depending on genotype, age, and gender. Both mouse models demonstrated concordant abundance changes of several genera influenced by one or more of the investigated factors. Abundance was significantly impacted by gender, highlighting the need to further elucidate the impact of gender differences. The findings underline the importance of the microbiome in PDAC development and indicate that microbiological screening of patients at risk and targeting the microbiome in PDAC development may be feasible in future.

CUX1 Enhances Pancreatic Cancer Formation by Synergizing with KRAS and Inducing MEK/ERK-Dependent Proliferation.

The transcription factor CUX1 has been implicated in either tumor suppression or progression, depending on the cancer entity and the prevalent CUX1 isoform. Previously, we could show that CUX1 acts as an important mediator of tumor cell proliferation and resistance to apoptosis in pancreatic cancer cell lines. However, in vivo evidence for its impact on pancreatic carcinogenesis, isoform-specific effects and downstream signaling cascades are missing. We crossbred two different CUX1 isoform mouse models (p200 CUX1 and p110 CUX1) with KC (KrasLSL-G12D/+; Ptf1aCre/+) mice, a genetic model for pancreatic precursor lesions (PanIN). In the context of oncogenic KRASs, both mice KCCux1p200 and KCCux1p110 led to increased PanIN formation and development of invasive pancreatic ductal adenocarcinomata (PDAC). In KCCux1p110 mice, tumor development was dramatically more accelerated, leading to formation of invasive PDAC within 4 weeks. In vitro and in vivo, we could show that CUX1 enhanced proliferation by activating MEK-ERK signaling via an upstream increase of ADAM17 protein, which in turn led to an activation of EGFR. Additionally, CUX1 further enhanced MEK-ERK activation through upregulation of the serine/threonine kinase MOS, phosphorylating MEK in a KRAS-independent manner. We identified p110 CUX1 as major driver of pancreatic cancer formation in the context of mutant KRAS. These results provide the first in vivo evidence for the importance of CUX1 in the development of pancreatic cancer, and highlight the importance of CUX1-dependent signaling pathways as potential therapeutic targets.

Correction: Interaction between somatostatin analogues and targeted therapies in neuroendocrine tumor cells.

[This corrects the article DOI: 10.1371/journal.pone.0218953.].

Interaction between somatostatin analogues and targeted therapies in neuroendocrine tumor cells.

Somatostatin analogues (SSA) represent the standard of care for symptom control in patients with functional gastro-entero-pancreatic neuroendocrine tumors (GEP-NET). In addition, SSA exert significant anti-proliferative effects in mid-gut and pancreatic NET (PanNET). In parallel, molecularly targeted therapies (MTT) have been shown to improve progression free survival (PFS) in patients with PanNET. However, due to either primary or acquired resistance to MTT, their impact on overall survival (OS) remains unclear. To date, various hypotheses exist to explain differences in patient responsiveness to SSA and MTT. However, data addressing one of the most pivotal questions, whether combining SSA with novel MTT will result in synergistic or additive efficacy compared to monotherapy, are lacking. The aim of this study is to characterize the interaction, optimal sequence and dosing of SSA-based and molecularly targeted therapies in PanNET. Somatostatin receptor subtypes 1-5 (SSTR) were evaluated in the neuroendocrine cell lines Bon1, QGP1 and Ins-1 via immunoblot and qRT-PCR. The impact of the SSA-analogue lanreotide alone or in combination with the MTT sunitinib, everolimus and regorafenib on intracellular signalling, hormone secretion and cell proliferation was determined in cell lysates and supernatants. In addition, synergistic effects of SSA and MTT in various sequential therapeutic approaches were investigated. SSTR were differently expressed in the examined neuroendocrine tumor cell lines. SSTR modulation via lanreotide moderately influenced proliferation, mainly via modulating AKT and ERK signalling, which was paralleled by decreased chromogranin A (CgA) expression and secretion. Interestingly, MTT treatment with regorafenib upregulated the expression of SSTR-2 and -5, while sunitinib and everolimus did not significantly alter SSTR expression. Cell viability was significantly reduced by all MTT, with regorafenib exerting the most significant effects. However, compared to the marked effects of MTT alone, synergistic effects of combined MTT and lanreotide treatment were only modest and time- and dose-dependent. SSTR are differentially expressed in various NEN cell lines. Their expression is influenced by MTT treatment. Various sequential or simultaneous combinations of lanreotide and MTT did not lead to significant synergistic effects.

Cellular and radiobiological effects of carbonic anhydrase IX in human breast cancer cells.

Hypoxia­induced carbonic anhydrase IX (CAIX) is involved in intracellular and extracellular pH regulation, which is critical for tumor growth and metastasis. CAIX is overexpressed in breast cancer and is associated with the poor survival of patients after radiotherapy. Therefore, we evaluated the cellular and radiobiological effects of CAIX inhibition in human breast cancer cells. We used CA9 siRNA and the CA inhibitor (CAI) U104, respectively, to inhibit CAIX expression and activity in basal triple­negative MDA­MB­231 and luminal MCF­7 cells under hypoxic conditions. We investigated the effects of CAIX inhibition on CA9 mRNA and CAIX protein level, as well as on CAIX activity, intracellular pH, proliferation, apoptosis, clonogenic survival, migration, cell cycle distribution and radiosensitivity. CA9 siRNA and CAI U104 decreased CA9 mRNA and CAIX protein level in MDA­MB­231 and MCF­7 cells. Furthermore, incubation with CAI U104 significantly decreased carbonic anhydrase activity and reduced the intracellular pH. Additionally, CA9 siRNA or U104 reduced clonogenic survival, migration and the number of cells in the G0/G1 phase, induced apoptosis and demonstrated additive or synergistic effects in combination with irradiation. In conclusion, combination of CAIX inhibition and irradiation is a promising treatment strategy against breast cancer with hypoxia­induced CAIX expression.

Betulinyl Sulfamates as Anticancer Agents and Radiosensitizers in Human Breast Cancer Cells.

Betulinic acid (BA), a natural compound of birch bark, is cytotoxic for many tumors. Recently, a betulinyl sulfamate was described that inhibits carbonic anhydrases (CA), such as CAIX, an attractive target for tumor-selective therapy strategies in hypoxic cancer cells. Data on combined CAIX inhibition with radiotherapy are rare. In the human breast cancer cell lines MDA-MB231 and MCF7, the effects of BA and betulinyl sulfamates on cellular and radiobiological behavior under normoxia and hypoxia were evaluated. The two most effective betulinyl sulfamates CAI 1 and CAI 3 demonstrated a 1.8-2.8-fold higher cytotoxicity than BA under normoxia in breast cancer cells, with IC50 values between 11.1 and 18.1 µM. BA exhibits its strongest cytotoxicity with IC50 values of 8.2 and 16.4 µM under hypoxia. All three substances show a dose-dependent increase in apoptosis, inhibition of migration, and inhibition of hypoxia-induced gene expression. In combination with irradiation, betulinyl sulfamates act as radiosensitizers, with DMF10 values of 1.47 (CAI 1) and 1.75 (CAI 3) under hypoxia in MDA-MB231 cells. BA showed additive effects in combination with irradiation. Taken together; our results suggest that BA and betulinyl sulfamates seem to be attractive substances to combine with radiotherapy; particularly for hypoxic breast cancer.