Mutant Kras- and p16-regulated NOX4 activation overcomes metabolic checkpoints in development of pancreatic ductal adenocarcinoma

Kras activation and p16 inactivation are required to develop pancreatic ductal adenocarcinoma (PDAC). However, the biochemical mechanisms underlying these double alterations remain unclear. Here we discover that NAD(P)H oxidase 4 (NOX4), an enzyme known to catalyse the oxidation of NAD(P)H, is upregulated when p16 is inactivated by looking at gene expression profiling studies. Activation of NOX4 requires catalytic subunit p22phox, which is upregulated following Kras activation. Both alterations are also detectable in PDAC cell lines and patient specimens. Furthermore, we show that elevated NOX4 activity accelerates oxidation of NADH and supports increased glycolysis by generating NAD+, a substrate for GAPDH-mediated glycolytic reaction, promoting PDAC cell growth. Mechanistically, NOX4 was induced through p16-Rb-regulated E2F and p22phox was induced by KrasG12V-activated NF-κB. In conclusion, we provide a biochemical explanation for the cooperation between p16 inactivation and Kras activation in PDAC development and suggest that NOX4 is a potential therapeutic target for PDAC

Nicotinamide nucleotide transhydrogenase-mediated redox homeostasis promotes tumor growth and metastasis in gastric cancer

Overcoming oxidative stress is a critical step for tumor growth and metastasis, however the underlying mechanisms in gastric cancer remain unclear. In this study, we found that overexpression of nicotinamide nucleotide transhydrogenase (NNT) was associated with shorter overall and disease free survival in gastric cancer. The NNT is considered a key antioxidative enzyme based on its ability to regenerate NADPH from NADH. Knockdown of NNT caused significantly NADPH reduction, induced high levels of ROS and significant cell apoptosis under oxidative stress conditions such as glucose deprival and anoikis. In vivo experiments showed that NNT promoted tumor growth, lung metastasis and peritoneal dissemination of gastric cancer. Moreover, intratumoral injection of NNT siRNA significantly suppressed gastric tumor growth in patient-derived xenograft (PDX) models. Overall, our study highlights the crucial functional roles of NNT in redox regulation and tumor progression and thus raises an important therapeutic hypothesis in gastric cancer. Keywords: NNT, Gastric cancer, NADPH, Anoikis resistance, Metastasi

Cooperativity of oncogenic K-ras and downregulated p16/INK4A in human pancreatic tumorigenesis.

Activation of K-ras and inactivation of p16 are the most frequently identified genetic alterations in human pancreatic epithelial adenocarcinoma (PDAC). Mouse models engineered with mutant K-ras and deleted p16 recapitulate key pathological features of PDAC. However, a human cell culture transformation model that recapitulates the human pancreatic molecular carcinogenesis is lacking. In this study, we investigated the role of p16 in hTERT-immortalized human pancreatic epithelial nestin-expressing (HPNE) cells expressing mutant K-ras (K-rasG12V). We found that expression of p16 was induced by oncogenic K-ras in these HPNE cells and that silencing of this induced p16 expression resulted in tumorigenic transformation and development of metastatic PDAC in an orthotopic xenograft mouse model. Our results revealed that PI3K/Akt, ERK1/2 pathways and TGFα signaling were activated by K-ras and involved in the malignant transformation of human pancreatic cells. Also, p38/MAPK pathway was involved in p16 up-regulation. Thus, our findings establish an experimental cell-based model for dissecting signaling pathways in the development of human PDAC. This model provides an important tool for studying the molecular basis of PDAC development and gaining insight into signaling mechanisms and potential new therapeutic targets for altered oncogenic signaling pathways in PDAC

Decreased TUSC3 Promotes Pancreatic Cancer Proliferation, Invasion and Metastasis

<div><p>Pancreatic cancer is an aggressive disease with dismal prognosis. It is of paramount importance to understand the underlying etiological mechanisms and identify novel, consistent, and easy-to-apply prognostic factors for precision therapy. TUSC3 (tumor suppressor candidate 3) was identified as a potential tumor suppressor gene and previous study showed TUSC3 is decreased in pancreatic cancer at mRNA level, but its putative tumor suppressor function remains to be verified. In this study, TUSC3 expression was found to be suppressed both at mRNA and protein levels in cell line models as well as in clinical samples; decreased TUSC3 expression was associated with higher pathological TNM staging and poorer outcome. In three pairs of cell lines with different NF-κB activity, TUSC3 expression was found to be reversely correlated with NF-κB activity. TUSC3-silenced pancreatic cancer cell line exhibited enhanced potential of proliferation, migration and invasion. In an orthotopic implanted mice model, TUSC3 silenced cells exhibited more aggressive phenotype with more liver metastasis. In conclusion, the current study shows that decreased immunological TUSC3 staining is a factor prognostic of poor survival in pancreatic cancer patients and decreased TUSC3 promotes pancreatic cancer cell proliferation, invasion and metastasis. The reverse correlation between NF-κB activity and TUSC3 expression may suggest a novel regulation pattern for this molecule.</p></div

Decreased TUSC3 Promotes Pancreatic Cancer Proliferation, Invasion and Metastasis - Fig 3

<p><b>TUSC3 is decreased in pancreatic cancer cell lines both at mRNA level (A) and protein level (B)</b>.</p

TUSC3 expression is associated with clinical pathological staging and Ki67 index.

<p>TUSC3 expression is associated with clinical pathological staging and Ki67 index.</p

Decreased TUSC3 expression promotes tumor cell growth, migration and invasion.

<p>(A, B)TUSC3 is effectively knocked-down in Colo357 cell lines shown by (A) RT-PCR and (B) Western blot. (C, D) Proliferation is enhanced with TUSC3 knockdown, cell number counts are significantly different at 72 hours (Colo357 TUSC3 shRNA2 vs Colo357 Scramble p = 0.0086, Colo357 TUSC3 shRNA3 vs Colo357 Scramble p = 0.0011) (D). (E, F) Colony Formation is enhanced with TSUC3 knockdown (Colo357 TUSC3 shRNA2 vs Colo357 Scramble p<0.0001, Colo357 TUSC3 shRNA3 vs Colo357 Scramble p<0.0001). (G) Migration Test(Colo357 TUSC3 shRNA2 vs Colo357 Scramble p<0.0001, Colo357 TUSC3 shRNA3 vs Colo357 Scramble p<0.0001). (H, I) Invasion Test (Colo357 TUSC3 shRNA2 vs Colo357 Scramble p<0.0001, Colo357 TUSC3 shRNA3 vs Colo357 Scramble p<0.0001). (J) Wound healing test showed more rapid closure of the gap in TUSC3 silenced cells. All experiments were performed three times with representative figures shown as above.</p

Univariate and Multivariate Cox Proportional Hazard Analysis of Factors Affecting Recurrence-free and Overall Survival in Pancreatic Cancer.

<p>Univariate and Multivariate Cox Proportional Hazard Analysis of Factors Affecting Recurrence-free and Overall Survival in Pancreatic Cancer.</p

TUSC3 is decreased in pancreatic cancer samples.

<p>(A) TUSC3 expression in non-neoplastic pancreatic tissues (acinar, duct and islets). (B) TUSC3 expression in pancreatic cancer cells. (C) Low TUSC3 expression in pancreatic cancer cells compared to islets. (D) Comparison of TUSC3 staining in primary PC and LN metastasis(paired t-test, p<0.001, n = 58). Original magnification×200.</p