Morgana acts as a proto‐oncogene through inhibition of a ROCK–PTEN pathway

Morgana/CHP‐1 is a ubiquitously expressed protein able to inhibit ROCK II kinase activity. We have previously demonstrated that morgana haploinsufficiency leads to multiple centrosomes, genomic instability, and higher susceptibility to tumour development. While a large fraction of human cancers has shown morgana down‐regulation, a small subset of tumours was shown to express high morgana levels. Here we demonstrate that high morgana expression in different breast cancer subtypes correlates with high tumour grade, mitosis number, and lymph node positivity. Moreover, morgana overexpression induces transformation in NIH‐3T3 cells and strongly protects them from various apoptotic stimuli. From a mechanistic point of view, we demonstrate that morgana causes PTEN destabilization, by inhibiting ROCK activity, hence triggering the PI3K/AKT survival pathway. In turn, morgana down‐regulation in breast cancer cells that express high morgana levels increases PTEN expression and leads to sensitization of cells to chemotherapy. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd     

Is a Preoperative Assessment of the Early Recurrence of Pancreatic Cancer Possible after Complete Surgical Resection?

Background/Aims

The prognosis of pancreatic adenocarcinoma (PAC) is poor. The serum carbohydrate antigen 19-9 (CA 19-9) level has been identified as a prognostic indicator of recurrence and reduced overall survival. The aim of this study was to identify preoperative prognostic factors and to create a prognostic model able to assess the early recurrence risk for patients with resectable PAC.

Methods

A series of 177 patients with PAC treated surgically at the St. Andrea Hospital of Rome between January 2003 and December 2011 were reviewed retrospectively. Univariate and multivariate analyses were utilized to identify preoperative prognostic indicators.

Results

A preoperative CA 19-9 level >228 U/mL, tumor size >3.1 cm, and the presence of pathological preoperative lymph nodes statistically correlated with early recurrence. Together, these three factors predicted the possibility of an early recurrence with 90.4% accuracy. The combination of these three preoperative conditions was identified as an independent parameter for early recurrence based on multivariate analysis (p=0.0314; hazard ratio, 3.9811; 95% confidence interval, 1.1745 to 15.3245).

Conclusions

PAC patient candidates for surgical resection should undergo an assessment of early recurrence risk to avoid unnecessary and ineffective resection and to identify patients for whom palliative or alternative treatment may be the treatment of choice.     

Genetic modifiers of EGFR dependence in non-small cell lung cancer

Lung adenocarcinomas harboring activating mutations in the epidermal growth factor receptor (EGFR) represent a common molecular subset of non-small cell lung cancer (NSCLC) cases. EGFR mutations predict sensitivity to EGFR tyrosine kinase inhibitors (TKIs) and thus represent a dependency in NSCLCs harboring these alterations, but the genetic basis of EGFR dependence is not fully understood. Here, we applied an unbiased, ORF-based screen to identify genetic modifiers of EGFR dependence in EGFR-mutant NSCLC cells. This approach identified 18 kinase and kinase-related genes whose overexpression can substitute for EGFR in EGFR-dependent PC9 cells, and these genes include seven of nine Src family kinase genes, FGFR1, FGFR2, ITK, NTRK1, NTRK2, MOS, MST1R, and RAF1. A subset of these genes can complement loss of EGFR activity across multiple EGFR-dependent models. Unbiased gene-expression profiling of cells overexpressing EGFR bypass genes, together with targeted validation studies, reveals EGFR-independent activation of the MEK-ERK and phosphoinositide 3-kinase (PI3K)-AKT pathways. Combined inhibition of PI3K-mTOR and MEK restores EGFR dependence in cells expressing each of the 18 EGFR bypass genes. Together, these data uncover a broad spectrum of kinases capable of overcoming dependence on EGFR and underscore their convergence on the PI3K-AKT and MEK-ERK signaling axes in sustaining EGFR-independent survival.The term “oncogene addiction” has been used to describe the phenomenon whereby tumor cells exhibit singular reliance on an oncogene or oncogenic pathway for their survival, despite the accumulation of multiple genetic lesions (1). In non-small cell lung cancer (NSCLC), this principle is perhaps best exemplified with the finding that epidermal growth factor receptor (EGFR) mutations predict response to EGFR tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib, and thus represent a dependency in the subset of tumors harboring these alterations (2–6). However, though EGFR-mutant NSCLCs typically respond dramatically to EGFR TKIs, clinical responses are not universal, even within this genetically defined cohort, with the rate of objective response estimated to be ∼71% (5, 6). Furthermore, the overwhelming majority of patients who initially respond to EGFR inhibitors ultimately develop resistance to therapy (7). A deeper understanding of the genetic underpinnings of EGFR addiction, and how EGFR-mutant cells can overcome reliance on EGFR, may improve clinical outcomes.Here, we have applied an unbiased screening approach to identify genetic modifiers of EGFR dependence in NSCLC. Mounting evidence supports the existence of several genetic modifiers of EGFR dependence in EGFR-mutant NSCLC, which can reduce the degree to which these tumors rely on EGFR and thereby contribute to EGFR TKI resistance (8). Examples include amplification of the MET receptor tyrosine kinase (RTK) (9), activation of the NF-κB pathway (8), amplification of the HER2 (ERBB2) RTK (10), amplification of the CRKL gene (11), and activation of the AXL kinase (12). Notably, MET bypass can be reciprocally achieved via EGFR activation in MET-dependent cells (13), and analogous examples of reciprocal kinase switching have been reported in other kinase-driven cancer models (14, 15). These and other findings suggest that compensatory kinase switching may be a more general way in which oncogene-dependent cancers overcome reliance on their primary driver kinase (14, 16), but the full-range of kinases capable of mediating EGFR bypass has not been systematically studied.Recent advances in large-scale functional genetic libraries have made it possible to query a wide range of genetic perturbations for their ability to modulate specific cellular phenotypes in mammalian systems (17, 18). Using the model of EGFR-mutant, erlotinib-sensitive NSCLC cells, we have performed a systematic ORF-based screen to identify kinase and kinase-related genes whose overexpression can complement loss of EGFR activity in an EGFR-dependent context. Our findings indicate broad potential for EGFR substitution in the setting of EGFR dependence, with compensatory mechanisms commonly conferring EGFR-independent activation of the PI3K-AKT and MEK-ERK signaling pathways. Importantly, this approach has recovered known mechanisms of erlotinib resistance as well as identified novel mediators of EGFR bypass in EGFR-mutant NSCLC. These data support the idea that the EGFR-dependent state can be redundantly driven by diverse genetic inputs that commonly converge on shared downstream signaling nodes.     

Enhanced activation of an amino-terminally truncated isoform of the voltage-gated proton channel HVCN1 enriched in malignant B cells

HVCN1 (Hydrogen voltage-gated channel 1) is the only mammalian voltage-gated proton channel. In human B lymphocytes, HVCN1 associates with the B-cell receptor (BCR) and is required for optimal BCR signaling and redox control. HVCN1 is expressed in malignant B cells that rely on BCR signaling, such as chronic lymphocytic leukemia (CLL) cells. However, little is known about its regulation in these cells. We found that HVCN1 was expressed in B cells as two protein isoforms. The shorter isoform (HVCN1_S) was enriched in B cells from a cohort of 76 CLL patients. When overexpressed in a B-cell lymphoma line, HVCN1_S responded more profoundly to protein kinase C-dependent phosphorylation. This more potent enhanced gating response was mediated by increased phosphorylation of the same residue responsible for enhanced gating in HVCN1_L, Thr²⁹. Furthermore, the association of HVCN1_S with the BCR was weaker, which resulted in its diminished internalization upon BCR stimulation. Finally, HVCN1_S conferred a proliferative and migratory advantage as well as enhanced BCR-dependent signaling. Overall, our data show for the first time, to our knowledge, the existence of a shorter isoform of HVCN1 with enhanced gating that is specifically enriched in malignant B cells. The properties of HVCN1_S suggest that it may contribute to the pathogenesis of BCR-dependent B-cell malignancies.The voltage-gated proton channel HVCN1 (or H_V1 or VSOP) is a small protein that conducts protons across membranes selectively (1, 2) and in a regulated manner. Previously, we described its function in B lymphocytes, where proton channels sustain B-cell receptor (BCR) signaling via regulation of reactive oxygen species production by the NADPH oxidase enzyme complex (3). In addition, we found HVCN1 to be directly associated with the BCR. Upon receptor stimulation, the BCR and HVCN1 were cointernalized to late endosomal/lysosomal organelles called “MIICs,” or MHC class II-containing compartments, where antigens bound to the BCR are digested into small peptides and loaded onto MHC class II molecules for presentation to T cells (3).HVCN1 is expressed not only by normal but also by malignant B cells, such as those in chronic lymphocytic leukemia (CLL) (3). CLL cells are characterized by their reliance on BCR signaling for survival and growth (4), so it is possible that they maintain or upregulate HVCN1 expression to sustain their growth. Other tumor cells, such as those in breast (5) and colorectal cancer (6), have been found to rely on HVCN1 for survival. In these tumor cells, proton channels prevent excessive acidification of the cytoplasm and allow increased cell migration. In malignant B cells, HVCN1 may regulate intracellular pH and at the same time sustain BCR signaling. However, its precise roles remain to be elucidated.We show here that CLL cells and other B-cell lines specifically express higher levels of a shorter isoform of HVCN1, HVCN1_S. We identified the existence of two distinct isoforms of relatively similar size when immunoblotting B-cell lysates with an HVCN1-specific antibody (3). HVCN1_S is only weakly expressed in normal B cells, and in light of its apparent upregulation in tumor cells, we set out to characterize its function. We show that HVCN1_S responds more strongly to phosphorylation by protein kinase C (PKC) and identify the phosphorylation site. We provide evidence that HVCN1_S in B cells is preferentially expressed at the plasma membrane, even upon BCR stimulation and subsequent internalization, due to a weaker association with the BCR. Finally, we show that HVCN1_S expression results in stronger BCR signaling, increased proliferation, and augmented chemokine-dependent migration. Overall, our data indicate that HVCN1_S is an alternative protein isoform that mediates stronger currents upon PKC phosphorylation, is more highly expressed at the plasma membrane, and can confer a growth advantage to malignant B cells.     

Prognosis and health-related quality of life in elderly patients after a mild to moderate trauma

The aims of this study were to compare outcome after a mild to moderate trauma in three subgroups of patients of increasing age (A1: <50, A2: 50–74, A3: >74 years) and to assess potential health-related quality of life (HRQOL) impairment. This is a follow-up study of a cohort of 418 trauma patients admitted to the High Dependency Unit of the Emergency Department of the University Hospital of Florence from July 2008 to February 2012. Six months after the event, a telephone interview using the Physical component summary (PCS) and Mental component summary (MCS) Health Composite Score (SF12) was conducted. Patients reported their QOL both at present and before trauma. In-hospital mortality was 10 of 418 (2.3 %); overall mortality was 27 of 244 (11 %) patients found at follow-up. No death was observed among A1 patients; overall mortality was (6/76) 7 % in A2 and (21/71) 30 % in A3 patients (p < 0.05 A1 vs A2, A1 vs A3 and A2 vs A3). Before the event, respectively, 94 and 96 % patients reported a normal MCS and PCS score (>39); after the event, the proportion of patients with a normal score value was significantly lower (MCS 70 %, p = 0.002; PCS 58 %, p < 0.0001). All subgroups showed a highly significant reduction in the scores’ value due to the trauma. After the event, the proportion of patients with normal scores was significantly lower (all p < 0.0001, except for MCS score in A1 subgroup, who showed p = 0.013) within all subgroups. Elderly patients’ prognosis was significantly worse compared with the younger counterpart; despite young patients’ optimal outcome, HRQOL was uniformly reduced across all age groups.