Dynamic interplay between structural variations and 3D chromosome organization in pancreatic cancer

Structural variations (SVs) are the greatest source of variation in the genome and can lead to oncogenesis. However, the identification and interpretation of SVs in human pancreatic cancer remain largely undefined due to technological limitations. Here, we investigate the spectrum of SVs and three-dimensional (3D) chromatin architecture in human pancreatic ductal epithelial cell carcinogenesis by using state-of-the-art long-read single-molecule real-time (SMRT) and high-throughput chromosome conformation capture (Hi-C) sequencing techniques. We find that the 3D genome organization is remodeled and correlated with gene expressional change. The bulk remodeling effect of cross-boundary SVs in the 3D genome partly depends on intercellular genomic heterogeneity. Meanwhile, contact domains tend to minimize these disrupting effects of SVs within local adjacent genomic regions to maintain overall stability of 3D genome organization. Moreover, our data also demonstrates complex genomic rearrangements involving two key driver genes CDKN2A and SMAD4, and elucidates their influence on cancer-related gene expression from both linear view and 3D perspective. Overall, this study provides a valuable resource and highlights the impact, complexity and dynamicity of the interplay between SVs and 3D genome organization, which further expands our understanding of pathogenesis of SVs in human pancreatic cancer.

Pancreatic ductal adenocarcinoma cells employ integrin α6β4 to form hemidesmosomes and regulate cell proliferation

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis due to its aggressive progression, late detection and lack of druggable driver mutations, which often combine to result in unsuitability for surgical intervention. Together with activating mutations of the small GTPase KRas, which are found in over 90% of PDAC tumours, a contributory factor for PDAC tumour progression is formation of a rigid extracellular matrix (ECM) and associated desmoplasia. This response leads to aberrant integrin signalling, and accelerated proliferation and invasion. To identify the integrin adhesion systems that operate in PDAC, we analysed a range of pancreatic ductal epithelial cell models using 2D, 3D and organoid culture systems. Proteomic analysis of isolated integrin receptor complexes from human pancreatic ductal epithelial (HPDE) cells predominantly identified integrin α6β4 and hemidesmosome components, rather than classical focal adhesion components. Electron microscopy, together with immunofluorescence, confirmed the formation of hemidesmosomes by HPDE cells, both in 2D and 3D culture systems. Similar results were obtained for the human PDAC cell line, SUIT-2. Analysis of HPDE cell secreted proteins and cell-derived matrices (CDM) demonstrated that HPDE cells secrete a range of laminin subunits and form a hemidesmosome-specific, laminin 332-enriched ECM. Expression of mutant KRas (G12V) did not affect hemidesmosome composition or formation by HPDE cells. Cell-ECM contacts formed by mouse and human PDAC organoids were also assessed by electron microscopy. Organoids generated from both the PDAC KPC mouse model and human patient-derived PDAC tissue displayed features of acinar-ductal cell polarity, and hemidesmosomes were visible proximal to prominent basement membranes. Furthermore, electron microscopy identified hemidesmosomes in normal human pancreas. Depletion of integrin β4 using siRNA reduced cell proliferation in both SUIT-2 and HPDE cells, reduced the number of SUIT-2 cells in S-phase, and induced G1 cell cycle arrest, indicating a requirement for α6β4-mediated adhesion for cell cycle progression and growth. Taken together, these data suggest that laminin-binding adhesion mechanisms in general, and hemidesmosome-mediated adhesion in particular, may be under-appreciated in the context of PDAC.

Relocation of phosphofructokinases within epithelial cells is a novel event preceding breast cancer recurrence that accurately predicts patient outcomes

Although recurrent cancers are often aggressive, little is known about the intracellular events required for cancer recurrences. Due to this lack of mechanistic information, there is no test to predict cancer recurrences or non-recurrences during early stages of disease. In this retrospective study, we use ductal carcinoma in situ (DCIS) of the breast as a framework to better understand the mechanism of cancer recurrences using patient outcomes as the physiological observable. Conventional pathology slides were labeled with anti-phosphofructokinase type L (PFKL) and anti-phosphofructokinase/fructose-2,6-bisphosphatase type 4 (PFKFB4) reagents. PFKL and PFKFB4 were found in ductal epithelial cell nucleoli from DCIS samples of women who did not experience a cancer recurrence. In contrast, PFKL and PFKFB4 may be found near the plasma membrane in samples from patients who will develop recurrent cancer. Using machine learning to predict patient outcomes, holdout studies of individual patient micrographs for the three biomarkers PFKL, PFKFB4, and phosphorylated GLUT1 demonstrated 38.6% true negatives, 49.5% true positives, 11.9% false positives and 0% false negatives (N=101). A sub-population of recurrent samples demonstrated PFKL, PFKFB4, and phosphorylated glucose transporter 1 accumulation at the apical surface of epithelial cells, suggesting that carbohydrates can be harvested from the ducts' luminal spaces as an energy source. We suggest that PFK isotype patterns are metabolic switches representing key mechanistic steps of recurrences. Furthermore, PFK enzyme patterns within epithelial cells contribute to an accurate diagnostic test to classify DCIS patients as high or low recurrence risk.

SRPX2 boosts pancreatic cancer chemoresistance by activating PI3K/AKT axis

Background and aimThis investigation was aimed at disclosing whether SRPX2 affected pancreatic cancer (PC) chemoresistance by regulating PI3K/Akt/mTOR signaling.MethodsTotally 243 PC patients were recruited, and they were incorporated into partial remission (PR) group, stable disease (SD) group and progressive disease (PD) group in accordance with their chemotherapeutic response. PC cell lines (i.e. AsPC1, Capan2, VFPAC-1, HPAC, PANC-1, BxPC-3 and SW1990) and human pancreatic ductal epithelial cell lines (hTERT-HPNE) were also collected.ResultsPC patients of SD + PD group were associated with higher post-chemotherapeutic SRPX2 level than PR group, and their post-chemotherapeutic SRPX2 level was above the pretherapeutic SRPX2 level (P < 0.05). PR population showed lower SRPX2 level after chemotherapy than before chemotherapy (P < 0.05). Besides high serum SRPX2 level and SRPX2 level change before and after chemotherapy were independent predictors of poor PC prognosis. Additionally, si-SRPX2 enhanced chemosensitivity of PC cell lines, and expressions of p-PI3K, p-AKT and p-mTOR were suppressed by si-SRPX2 (P < 0.05). IGF-1 treatment could changeover the impact of si-SRPX2 on proliferation, migration, invasion and chemoresistance of PC cells (P < 0.05).ConclusionThe SRPX2-PI3K/AKT/mTOR axis could play a role in modifying progression and chemoresistance of PC cells, which might help to improve PC prognosis.

Upregulation of DAB2IP Inhibits Ras Activity and Tumorigenesis in Human Pancreatic Cancer Cells

KRAS mutation-induced Ras activation plays an important role in the pathogenesis of pancreatic cancer, but the role of wild-type Ras and Ras GTPase-activating proteins remains unclear. The present study was designed to determine the expression spectra of Ras GTPase-activating proteins genes in pancreatic cancer cells, and the role of DAB2IP, a Ras GTPase-activating proteins gene, in the development and progression of pancreatic cancer. Following the analyses of the expression profiles of 16 Ras GTPase-activating proteins in 6 pancreatic cancer cell lines including Bxpc-3 (with wild-type KRAS), Capan-2, Sw1990, Aspc-1, CFPAC-1, and Panc-1 (with mutant KRAS) and 1 normal human pancreatic ductal epithelial cell line, H6C7, the expression of DAB2IP messenger RNA was further analyzed by quantitative real-time polymerase chain reaction. The role of DAB2IP in pancreatic cancer was further investigated in vitro and in vivo by upregulating DAB2IP in Bxpc-3 cells through transfection of DAB2IP into Bxpc-3 cells with recombinant lentivirus. The DAB2IP expression in pancreatic cancer cells and tissues with wild-type KRAS was significantly lower than that in cells and tissues with mutant KRAS ( P < .05). In Bxpc-3 cells with wild-type KRAS, overexpression of DAB2IP decreased the expression of P-AKT and P-ERK and the Ras activity; increased the expression of P-JNK and caspase 3; inhibited cell proliferation, invasiveness, and migration; and increased the cell sensitivity to cetuximab. Overexpression of DAB2IP inhibited tumor progression in a mouse model. In conclusion, DAB2IP downregulates Ras activity in wild-type pancreatic cancer cells. Overexpression of DAB2IP decreases the Ras activity, inhibits cell proliferation, and increases sensitivity to cetuximab in wild-type pancreatic cancer cells. In conclusion, DAB2IP may serve as a potential molecular therapeutic target for the treatment of pancreatic cancer.