TGF-ß Targets the Hippo Pathway Scaffold RASSF1A to Facilitate YAP/SMAD2 Nuclear Translocation.

Epigenetic inactivation of the Hippo pathway scaffold RASSF1A is associated with poor prognosis in a wide range of sporadic human cancers. Loss of expression reduces tumor suppressor activity and promotes genomic instability, but how this pleiotropic biomarker is regulated at the protein level is unknown. Here we show that TGF-ß is the physiological signal that stimulates RASSF1A degradation by the ubiquitin-proteasome pathway. In response to TGF-ß, RASSF1A is recruited to TGF-ß receptor I and targeted for degradation by the co-recruited E3 ubiquitin ligase ITCH. RASSF1A degradation is necessary to permit Hippo pathway effector YAP1 association with SMADs and subsequent nuclear translocation of receptor-activated SMAD2. We find that RASSF1A expression regulates TGF-ß-induced YAP1/SMAD2 interaction and leads to SMAD2 cytoplasmic retention and inefficient transcription of TGF-ß targets genes. Moreover, RASSF1A limits TGF-ß induced invasion, offering a new framework on how RASSF1A affects YAP1 transcriptional output and elicits its tumor-suppressive function.

TET2 Drives 5hmc Marking of GATA6 and Epigenetically Defines Pancreatic Ductal Adenocarcinoma Transcriptional Subtypes.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is characterized by advanced disease stage at presentation, aggressive disease biology, and resistance to therapy, resulting in an extremely poor 5-year survival rate of <10%. PDAC is classified into transcriptional subtypes with distinct survival characteristics, although how these arise is not known. Epigenetic deregulation, rather than genetics, has been proposed to underpin progression, but exactly why is unclear and is hindered by the technical limitations of analyzing clinical samples. METHODS: We performed genome-wide epigenetic mapping of DNA modifications 5-methylcytosine and 5-hydroxymethylcytosine (5hmc) using oxidative bisulfite sequencing from formalin-embedded sections. We identified overlap with transcriptional signatures in formalin-fixed, paraffin-embedded tissue from resected patients, via bioinformatics using iCluster and mutational profiling and confirmed them in vivo. RESULTS: We found that aggressive squamous-like PDAC subtypes result from epigenetic inactivation of loci, including GATA6, which promote differentiated classical pancreatic subtypes. We showed that squamous-like PDAC transcriptional subtypes are associated with greater loss of 5hmc due to reduced expression of the 5-methylcytosine hydroxylase TET2. Furthermore, we found that SMAD4 directly supports TET2 levels in classical pancreatic tumors, and loss of SMAD4 expression was associated with reduced 5hmc, GATA6, and squamous-like tumors. Importantly, enhancing TET2 stability using metformin and vitamin C/ascorbic acid restores 5hmc and GATA6 levels, reverting squamous-like tumor phenotypes and WNT-dependence in vitro and in vivo. CONCLUSIONS: We identified epigenetic deregulation of pancreatic differentiation as an underpinning event behind the emergence of transcriptomic subtypes in PDAC. Our data showed that restoring epigenetic control increases biomarkers of classical pancreatic tumors that are associated with improved therapeutic responses and survival.

Circulating biomarkers and outcomes from a randomised phase 2 trial of gemcitabine versus capecitabine-based chemoradiotherapy for pancreatic cancer.

BACKGROUND: The Phase 2 SCALOP trial compared gemcitabine with capecitabine-based consolidation chemoradiotherapy (CRT) in locally advanced pancreatic cancer (LAPC). METHODS: Thirty-five systematically identified circulating biomarkers were analysed in plasma samples from 60 patients enroled in SCALOP. Each was measured in triplicate at baseline (prior to three cycles of gemcitabine-capecitabine induction chemotherapy) and, for a subset, prior to CRT. Association with overall survival (OS) was determined using univariable Cox regression and optimal thresholds delineating low to high values identified using time-dependent ROC curves. Independence from known prognostic factors was assessed using Spearman correlation and the Wilcoxon rank sum test prior to multivariable Cox regression modelling including independent biomarkers and known prognostic factors. RESULTS: Baseline circulating levels of C-C motif chemokine ligand 5 (CCL5) were significantly associated with OS, independent of other clinicopathological characteristics. Patients with low circulating CCL5 (CCL5low) had a median OS of 18.5 (95% CI 11.76-21.32) months compared to 11.3 (95% CI 9.86-15.51) months in CCL5high; hazard ratio 1.95 (95% CI 1.04-8.65; p = 0.037). CONCLUSIONS: CCL5 is an independent prognostic biomarker in LAPC. Given the known role of CCL5 in tumour invasion, metastasis and the induction of an immunosuppressive micro-environment, targeting of CCL5-mediated pathways may offer therapeutic potential in pancreatic cancer. CLINICAL TRIAL REGISTRATION: The SCALOP trial was registered with ISRCTN, number 96169987 (registered 29 May 2008).

A Novel Composite Biomarker Panel For Detection Of Early Stage Non-small Cell Lung Cancer.

PURPOSE: To investigate a novel composite methodology of using targeted serum microRNAs (micro ribonucleic acid; miRNA) and urine metabolites for the accurate detection of early stage non-small cell lung cancer (NSCLC). METHODS: Consecutively consenting NSCLC patients and matched control subjects were recruited to provide samples of serum for miRNA and/or urine for metabolite analyses. Serum miRNA levels were measured using quantitative real-time reverse-transcription with exogenous control, and the comparative delta cycle threshold (CT) method was used to calculate relative miRNA expression of two targeted miRNAs (miR-21 and miR-223). The concentrations of six targeted urinary metabolites in patients and healthy controls were measured using proton nuclear magnetic resonance (1H NMR) spectroscopy. A composite methodology of using the 35 accruals with both serum and urine biomarkers was then established with binary logistic regression, receiver operating characteristic (ROC) models with or without artificial intelligence (AI). RESULTS: The ROC analysis of miRNA expression yielded a sensitivity of 96.4% and a specificity of 88.2% for the detection of early stage NSCLC, with area under the curve (AUC) = 0.91 (CI 95%: 0.80-1.0). Relative urinary concentrations of 4-methoxyphenylacetic acid (4MPLA) were significantly different between NSCLC and healthy control (p=0.008). The ROC analysis of 4MPLA yielded a sensitivity of 82.1% and a specificity of 88.2%, with AUC = 0.85. The composite process combining miRNA and metabolite expression demonstrated a sensitivity and specificity of nearly 100% and AUC=1. CONCLUSIONS: A highly specific, sensitive and non-invasive detection method for NSCLC was developed. Pending validation, this can potentially improve the early detection and, hence, the treatment and survival outcomes of patients.

Recurrent radiation recall dermatitis 40 years after radiation therapy for breast cancer.

Radiation recall dermatitis is an inflammatory reaction of the skin, which occurs at previously irradiated areas, usually following a subsequent exposure to an aggravating factor. Recall dermatitis can occur weeks to months after radiation, and the longest duration between radiation and dermatitis has been reported to be about 25 years. Here, we report a case of recurrent radiation recall dermatitis that developed spontaneously after 40 years following radiation for breast cancer. This case suggests that radiation recall dermatitis can occur much later than previously reported. In spite of this late presentation, topical anti-inflammatory agents managed the condition well.

PI3K/Akt-mediated regulation of p53 in cancer.

Mutations activating the PI3K (phosphoinositide 3-kinase)/Akt signalling pathway and inactivating the TP53 tumour-suppressor gene are common mechanisms that cancer cells require to proliferate and escape pre-programmed cell death. In a well-described mechanism, Akt mediates negative control of p53 levels through enhancing MDM2 (murine double minute 2)-mediated targeting of p53 for degradation. Accumulating evidence is beginning to suggest that, in certain circumstances, PTEN (phosphatase and tensin homologue deleted on chromosome 10)/PI3K/Akt also promotes p53 translation and protein stability, suggesting that additional mechanisms may be involved in the Akt-mediated regulation of p53 in tumours. In the present article, we discuss these aspects in the light of clinical PI3K/Akt inhibitors, where information regarding the effect on p53 activity will be a crucial factor that will undoubtedly influence therapeutic efficacy.

Concomitant Use of Proton Pump Inhibitors With Capecitabine Based Neoadjuvant Chemoradiotherapy for Locally Advanced Rectal Cancer: Is it Safe?

AIM: Capecitabine (Cape) is routinely used for the neoadjuvant chemoradiation treatment (NACRT) of locally advanced rectal cancers (LARCs). Previous reports have suggested that the concomitant use of proton pump inhibitors (PPIs) may affect the efficacy of Cape, although the true effect of PPIs when used with Cape as a radiosensitizer for neoadjuvant radiation is unclear. The aim of our study was to evaluate the impact of concurrent PPI use along with fluorouracil (FU) and Cape based NACRT in terms of pathologic and oncological outcomes, in patients with LARC. METHODS: LARC patients treated at our center with NACRT from 2010 to 2016 were identified. Postoperative pathology and follow-up outcomes were examined for any differences with relation to the use of PPIs concurrently with FU and Cape based NACRT and adjuvant chemotherapy regimens. RESULTS: Three hundred four and 204 patients received treatment with FU and Cape based NACRT. No difference in pathologic complete response rate was noted between the 2 arms with the concurrent use of PPIs (25.8% and 25%, respectively, P=0.633); or with and without the use of PPIs in the Cape-NACRT arm specifically (20% and 20.7%, P=0.945). At a median follow-up of 5 years, no statistical difference in local or distant control was noted in the Cape-NACRT patients, with and without concomitant PPI use (P=0.411 and 0.264, respectively).Multivariate analysis showed no association of PPI use and NACRT with Cape, in terms of local control (hazard ratio=0.001, P=0.988) or overall survival (hazard ratio=1.179, confidence interval=0.249-5.579, P=0.835). CONCLUSIONS: Our study revealed that there was no adverse pathologic or oncological outcome with the concurrent use of PPIs along with Cape-NACRT in the treatment of LARC. We report that it may be safe to use PPIs if essential, in this clinical setting, although it would be wise to exercise caution.

Breast Cancer Care in South India: Is Practice Concordant With National Guidelines?

PURPOSE: The National Cancer Grid (NCG) of India has recently published clinical practice guidelines that are relevant in the Indian context. We evaluated the extent to which breast cancer care at a teaching hospital in South India was concordant with NCG guidelines. METHODS: All patients who had surgery for breast cancer at a single center from January 2014 to December 2015 were included. Demographic, pathologic, and treatment characteristics were extracted from the electronic medical record. Patients were classified as being concordant with six elements selected from the NCG guideline. The indicators related to appropriate use of sentinel lymph node (SLN) biopsy, lymph node harvest, adjuvant radiotherapy, adjuvant chemotherapy, human epidermal growth factor receptor 2 (HER2) testing, and delivery of adjuvant trastuzumab. RESULTS: A total of 401 women underwent surgery for breast cancer; mean age (standard deviation) was 57 (12) years. Lymph node involvement was present in 47% (188 of 401) of the cohort; 23% (94 of 401) had T1 disease. Ninety-two percent (368 of 401) underwent radical modified mastectomy. SLN biopsy was performed in 75% (167 of 222) of eligible patients. Eighty percent (208 of 261) of patients with a positive SLN biopsy or no SLN biopsy had a lymph node harvest of more than 10. Adjuvant chemotherapy with an anthracycline and a taxane was delivered to 67% of patients (118 of 177) with node-positive disease. Adjuvant radiotherapy was delivered to 84% (180 of 213) of patients with breast-conserving surgery, T4 tumors, or 3+ positive lymph nodes. Fluorescent in situ hybridization testing was performed in 59% of patients (43 of 73) with 2+ HER2-positive lymph nodes on immunohistochemistry. Among patients with HER2 overexpression, 40% (36 of 91) received adjuvant trastuzumab. CONCLUSION: Concordance with NCG guidelines for breast cancer care ranged from 40% to 84%. Guideline concordance was lowest for those elements of care associated with the highest direct costs to patients.

ARCII: A phase II trial of the HIV protease inhibitor Nelfinavir in combination with chemoradiation for locally advanced inoperable pancreatic cancer.

BACKGROUND AND PURPOSE: Nelfinavir can enhance intrinsic radiosensitivity, reduce hypoxia and improve vascularity. We conducted a phase II trial combining nelfinavir with chemoradiotherapy (CRT) for locally advanced inoperable pancreatic cancer (LAPC). MATERIALS AND METHODS: Radiotherapy (50.4Gy/28 fractions; boost to 59.4Gy/33 fractions) was administered with weekly gemcitabine and cisplatin. Nelfinavir started 3-10days before and was continued during CRT. The primary end-point was 1-year overall survival (OS). Secondary end-points included histological downstaging, radiological response, 1-year progression free survival (PFS), overall survival (OS) and treatment toxicity. An imaging sub-study (n=6) evaluated hypoxia ((18)F-Fluoromisonidazole-PET) and perfusion (perfusion CT) during induction nelfinavir. RESULTS: The study closed after recruiting 23 patients, due to non-availability of Nelfinavir in Europe. The 1-year OS was 73.4% (90% CI: 54.5-85.5%) and median OS was 17.4months (90% CI: 12.8-18.8). The 1-year PFS was 21.8% (90% CI: 8.9-38.3%) and median PFS was 5.5months (90% CI: 4.1-8.3). All patients experienced Grade 3/4 toxicity, but many were asymptomatic laboratory abnormalities. Four of 6 patients on the imaging sub-study demonstrated reduced hypoxia and increased perfusion post-nelfinavir. CONCLUSIONS: CRT combined with nelfinavir showed acceptable toxicity and promising survival in pancreatic cancer.

Hypoxia-induced p53 modulates both apoptosis and radiosensitivity via AKT.

Restoration of hypoxia-induced apoptosis in tumors harboring p53 mutations has been proposed as a potential therapeutic strategy; however, the transcriptional targets that mediate hypoxia-induced p53-dependent apoptosis remain elusive. Here, we demonstrated that hypoxia-induced p53-dependent apoptosis is reliant on the DNA-binding and transactivation domains of p53 but not on the acetylation sites K120 and K164, which, in contrast, are essential for DNA damage-induced, p53-dependent apoptosis. Evaluation of hypoxia-induced transcripts in multiple cell lines identified a group of genes that are hypoxia-inducible proapoptotic targets of p53, including inositol polyphosphate-5-phosphatase (INPP5D), pleckstrin domain-containing A3 (PHLDA3), sulfatase 2 (SULF2), B cell translocation gene 2 (BTG2), cytoplasmic FMR1-interacting protein 2 (CYFIP2), and KN motif and ankyrin repeat domains 3 (KANK3). These targets were also regulated by p53 in human cancers, including breast, brain, colorectal, kidney, bladder, and melanoma cancers. Downregulation of these hypoxia-inducible targets associated with poor prognosis, suggesting that hypoxia-induced apoptosis contributes to p53-mediated tumor suppression and treatment response. Induction of p53 targets, PHLDA3, and a specific INPP5D transcript mediated apoptosis in response to hypoxia through AKT inhibition. Moreover, pharmacological inhibition of AKT led to apoptosis in the hypoxic regions of p53-deficient tumors and consequently increased radiosensitivity. Together, these results identify mediators of hypoxia-induced p53-dependent apoptosis and suggest AKT inhibition may improve radiotherapy response in p53-deficient tumors.

AKT regulates NPM dependent ARF localization and p53mut stability in tumors.

Nucleophosmin (NPM) is known to regulate ARF subcellular localization and MDM2 activity in response to oncogenic stress, though the precise mechanism has remained elusive. Here we describe how NPM and ARF associate in the nucleoplasm to form a MDM2 inhibitory complex. We find that oligomerization of NPM drives nucleolar accumulation of ARF. Moreover, the formation of NPM and ARF oligomers antagonizes MDM2 association with the inhibitory complex, leading to activation of MDM2 E3-ligase activity and targeting of p53. We find that AKT phosphorylation of NPM-Ser48 prevents oligomerization that results in nucleoplasmic localization of ARF, constitutive MDM2 inhibition and stabilization of p53. We also show that ARF promotes p53 mutant stability in tumors and suppresses p73 mediated p21 expression and senescence. We demonstrate that AKT and PI3K inhibitors may be effective in treatment of therapeutically resistant tumors with elevated AKT and carrying gain of function mutations in p53. Our results show that the clinical candidate AKT inhibitor MK-2206 promotes ARF nucleolar localization, reduced p53(mut) stability and increased sensitivity to ionizing radiation in a xenograft model of pancreatic cancer. Analysis of human tumors indicates that phospho-S48-NPM may be a useful biomarker for monitoring AKT activity and in vivo efficacy of AKT inhibitor treatment. Critically, we propose that combination therapy involving PI3K-AKT inhibitors would benefit from a patient stratification rationale based on ARF and p53(mut) status.

Menin determines K-RAS proliferative outputs in endocrine cells.

Endocrine cell proliferation fluctuates dramatically in response to signals that communicate hormone demand. The genetic alterations that override these controls in endocrine tumors often are not associated with oncogenes common to other tumor types, suggesting that unique pathways govern endocrine proliferation. Within the pancreas, for example, activating mutations of the prototypical oncogene KRAS drive proliferation in all pancreatic ductal adenocarcimomas but are never found in pancreatic endocrine tumors. Therefore, we asked how cellular context impacts K-RAS signaling. We found that K-RAS paradoxically suppressed, rather than promoted, growth in pancreatic endocrine cells. Inhibition of proliferation by K-RAS depended on antiproliferative RAS effector RASSF1A and blockade of the RAS-activated proproliferative RAF/MAPK pathway by tumor suppressor menin. Consistent with this model, a glucagon-like peptide 1 (GLP1) agonist, which stimulates ERK1/2 phosphorylation, did not affect endocrine cell proliferation by itself, but synergistically enhanced proliferation when combined with a menin inhibitor. In contrast, inhibition of MAPK signaling created a synthetic lethal interaction in the setting of menin loss. These insights suggest potential strategies both for regenerating pancreatic ß cells for people with diabetes and for targeting menin-sensitive endocrine tumors.