PR55α-controlled protein phosphatase 2A inhibits p16 expression and blocks cellular senescence induction by γ-irradiation.

Cellular senescence is a permanent cell cycle arrest that can be triggered by both internal and external genotoxic stressors, such as telomere dysfunction and DNA damage. The execution of senescence is mainly by two pathways, p16/RB and p53/p21, which lead to CDK4/6 inhibition and RB activation to block cell cycle progression. While the regulation of p53/p21 signaling in response to DNA damage and other insults is well-defined, the regulation of the p16/RB pathway in response to various stressors remains poorly understood. Here, we report a novel function of PR55α, a regulatory subunit of PP2A Ser/Thr phosphatase, as a potent inhibitor of p16 expression and senescence induction by ionizing radiation (IR), such as γ-rays. The results show that ectopic PR55α expression in normal pancreatic cells inhibits p16 transcription, increases RB phosphorylation, and blocks IR-induced senescence. Conversely, PR55α-knockdown by shRNA in pancreatic cancer cells elevates p16 transcription, reduces RB phosphorylation, and triggers senescence induction after IR. Furthermore, this PR55α function in the regulation of p16 and senescence is p53-independent because it was unaffected by the mutational status of p53. Moreover, PR55α only affects p16 expression but not p14 (ARF) expression, which is also transcribed from the same CDKN2A locus but from an alternative promoter. In normal human tissues, levels of p16 and PR55α proteins were inversely correlated and mutually exclusive. Collectively, these results describe a novel function of PR55α/PP2A in blocking p16/RB signaling and IR-induced cellular senescence.

Cell Signaling Pathways That Promote Radioresistance of Cancer Cells.

Radiation therapy (RT) is a standard treatment for solid tumors and about 50% of patients with cancer, including pediatric cancer, receive RT. While RT has significantly improved the overall survival and quality of life of cancer patients, its efficacy has still been markedly limited by radioresistance in a significant number of cancer patients (intrinsic or acquired), resulting in failure of the RT control of the disease. Radiation eradicates cancer cells mainly by causing DNA damage. However, radiation also concomitantly activates multiple prosurvival signaling pathways, which include those mediated by ATM, ATR, AKT, ERK, and NF-κB that promote DNA damage checkpoint activation/DNA repair, autophagy induction, and/or inhibition of apoptosis. Furthermore, emerging data support the role of YAP signaling in promoting the intrinsic radioresistance of cancer cells, which occurs through its activation of the transcription of many essential genes that support cell survival, DNA repair, proliferation, and the stemness of cancer stem cells. Together, these signaling pathways protect cancer cells by reducing the magnitude of radiation-induced cytotoxicity and promoting radioresistance. Thus, targeting these prosurvival signaling pathways could potentially improve the radiosensitivity of cancer cells. In this review, we summarize the contribution of these pathways to the radioresistance of cancer cells.

The GSK3 kinase and LZTR1 protein regulate the stability of Ras family proteins and the proliferation of pancreatic cancer cells.

Ras family proteins are membrane-bound GTPases that control proliferation, survival, and motility. Many forms of cancers are driven by the acquisition of somatic mutations in a RAS gene. In pancreatic cancer (PC), more than 90% of tumors carry an activating mutation in KRAS. Mutations in components of the Ras signaling pathway can also be the cause of RASopathies, a group of developmental disorders. In a subset of RASopathies, the causal mutations are in the LZTR1 protein, a substrate adaptor for E3 ubiquitin ligases that promote the degradation of Ras proteins. Here, we show that the function of LZTR1 is regulated by the glycogen synthase kinase 3 (GSK3). In PC cells, inhibiting or silencing GSK3 led to a decline in the level of Ras proteins, including both wild type Ras proteins and the oncogenic Kras protein. This decline was accompanied by a 3-fold decrease in the half-life of Ras proteins and was blocked by the inhibition of the proteasome or the knockdown of LZTR1. Irrespective of the mutational status of KRAS, the decline in Ras proteins was observed and accompanied by a loss of cell proliferation. This loss of proliferation was blocked by the knockdown of LZTR1 and could be recapitulated by the silencing of either KRAS or GSK3. These results reveal a novel GSK3-regulated LZTR1-dependent mechanism that controls the stability of Ras proteins and proliferation of PC cells. The significance of this novel pathway to Ras signaling and its contribution to the therapeutic properties of GSK3 inhibitors are both discussed.

Ecdysoneless Protein Regulates Viral and Cellular mRNA Splicing to Promote Cervical Oncogenesis.

High-risk human papillomaviruses (HPV), exemplified by HPV16/18, are causally linked to human cancers of the anogenital tract, skin, and upper aerodigestive tract. Previously, we identified Ecdysoneless (ECD) protein, the human homolog of the Drosophila ecdysoneless gene, as a novel HPV16 E6-interacting protein. Here, we show that ECD, through its C-terminal region, selectively binds to high-risk but not to low-risk HPV E6 proteins. We demonstrate that ECD is overexpressed in cervical and head and neck squamous cell carcinoma (HNSCC) cell lines as well as in tumor tissues. Using The Cancer Genome Atlas dataset, we show that ECD mRNA overexpression predicts shorter survival in patients with cervical and HNSCC. We demonstrate that ECD knockdown in cervical cancer cell lines led to impaired oncogenic behavior, and ECD co-overexpression with E7 immortalized primary human keratinocytes. RNA-sequencing analyses of SiHa cells upon ECD knockdown showed to aberrations in E6/E7 RNA splicing, as well as RNA splicing of several HPV oncogenesis-linked cellular genes, including splicing of components of mRNA splicing machinery itself. Taken together, our results support a novel role of ECD in viral and cellular mRNA splicing to support HPV-driven oncogenesis. IMPLICATIONS: This study links ECD overexpression to poor prognosis and shorter survival in HNSCC and cervical cancers and identifies a critical role of ECD in cervical oncogenesis through regulation of viral and cellular mRNA splicing.

Disruption of FDPS/Rac1 axis radiosensitizes pancreatic ductal adenocarcinoma by attenuating DNA damage response and immunosuppressive signalling.

BACKGROUND: Radiation therapy (RT) has a suboptimal effect in patients with pancreatic ductal adenocarcinoma (PDAC) due to intrinsic and acquired radioresistance (RR). Comprehensive bioinformatics and microarray analysis revealed that cholesterol biosynthesis (CBS) is involved in the RR of PDAC. We now tested the inhibition of the CBS pathway enzyme, farnesyl diphosphate synthase (FDPS), by zoledronic acid (Zol) to enhance radiation and activate immune cells. METHODS: We investigated the role of FDPS in PDAC RR using the following methods: in vitro cell-based assay, immunohistochemistry, immunofluorescence, immunoblot, cell-based cholesterol assay, RNA sequencing, tumouroids (KPC-murine and PDAC patient-derived), orthotopic models, and PDAC patient's clinical study. FINDINGS: FDPS overexpression in PDAC tissues and cells (P < 0.01 and P < 0.05) is associated with poor RT response and survival (P = 0.024). CRISPR/Cas9 and pharmacological inhibition (Zol) of FDPS in human and mouse syngeneic PDAC cells in conjunction with RT conferred higher PDAC radiosensitivity in vitro (P < 0.05, P < 0.01, and P < 0.001) and in vivo (P < 0.05). Interestingly, murine (P = 0.01) and human (P = 0.0159) tumouroids treated with Zol+RT showed a significant growth reduction. Mechanistically, RNA-Seq analysis of the PDAC xenografts and patients-PBMCs revealed that Zol exerts radiosensitization by affecting Rac1 and Rho prenylation, thereby modulating DNA damage and radiation response signalling along with improved systemic immune cells activation. An ongoing phase I/II trial (NCT03073785) showed improved failure-free survival (FFS), enhanced immune cell activation, and decreased microenvironment-related genes upon Zol+RT treatment. INTERPRETATION: Our findings suggest that FDPS is a novel radiosensitization target for PDAC therapy. This study also provides a rationale to utilize Zol as a potential radiosensitizer and as an immunomodulator in PDAC and other cancers. FUNDING: National Institutes of Health (P50, P01, and R01).

p53/FBXL20 axis negatively regulates the protein stability of PR55α, a regulatory subunit of PP2A Ser/Thr phosphatase.

We have previously reported an important role of PR55α, a regulatory subunit of PP2A Ser/Thr phosphatase, in the support of critical oncogenic pathways required for oncogenesis and the malignant phenotype of pancreatic cancer. The studies in this report reveal a novel mechanism by which the p53 tumor suppressor inhibits the protein-stability of PR55α via FBXL20, a p53-target gene that serves as a substrate recognition component of the SCF (Skp1_Cullin1_F-box) E3 ubiquitin ligase complex that promotes proteasomal degradation of its targeted proteins. Our studies show that inactivation of p53 by siRNA-knockdown, gene-deletion, HPV-E6-mediated degradation, or expression of the loss-of-function mutant p53R175H results in increased PR55α protein stability, which is accompanied by reduced protein expression of FBXL20 and decreased ubiquitination of PR55α. Subsequent studies demonstrate that knockdown of FBXL20 by siRNA mimics p53 deficiency, reducing PR55α ubiquitination and increasing PR55α protein stability. Functional tests indicate that ectopic p53R175H or PR55α expression results in an increase of c-Myc protein stability with concomitant dephosphorylation of c-Myc-T58, which is a PR55α substrate, whose phosphorylation otherwise promotes c-Myc degradation. A significant increase in anchorage-independent proliferation is also observed in normal human pancreatic cells expressing p53R175H or, to a greater extent, overexpressing PR55α. Consistent with the common loss of p53 function in pancreatic cancer, FBXL20 mRNA expression is significantly lower in pancreatic cancer tissues compared to pancreatic normal tissues and low FBXL20 levels correlate with poor patient survival. Collectively, these studies delineate a novel mechanism by which the p53/FBXL20 axis negatively regulates PR55α protein stability.

Small molecule binding to inhibitor of nuclear factor kappa-B kinase subunit beta in an ATP non-competitive manner.

Inhibitor of nuclear factor kappa-B kinase subunit beta (IKKß) is a key regulator of the cannonical NF-κB pathway. IKKß has been validated as a drug target for pathological conditions, which include chronic inflammatory diseases and cancer. Pharmacological studies revealed that chronic administration of ATP-competitive IKKß inhibitors resulted in unexpected toxicity. We previously reported the discovery of 13-197 as a non-toxic IKKß inhibitor that reduced tumor growth. Here, we show that 13-197 inhibits IKKß in a ATP non-competitive manner and an allosteric pocket at the interface of the kinase and ubiquitin like domains was identified as the potential binding site.

Correction: Inhibition of RAC1 GTPase sensitizes pancreatic cancer cells to γ-irradiation.

[This corrects the article DOI: 10.18632/oncotarget.2500.].

PR55α regulatory subunit of PP2A inhibits the MOB1/LATS cascade and activates YAP in pancreatic cancer cells.

PP2A holoenzyme complexes are responsible for the majority of Ser/Thr phosphatase activities in human cells. Each PP2A consists of a catalytic subunit (C), a scaffold subunit (A), and a regulatory subunit (B). While the A and C subunits each exists only in two highly conserved isoforms, a large number of B subunits share no homology, which determines PP2A substrate specificity and cellular localization. It is anticipated that different PP2A holoenzymes play distinct roles in cellular signaling networks, whereas PP2A has only generally been defined as a putative tumor suppressor, which is mostly based on the loss-of-function studies using pharmacological or biological inhibitors for the highly conserved A or C subunit of PP2A. Recent studies of specific pathways indicate that some PP2A complexes also possess tumor-promoting functions. We have previously reported an essential role of PR55α, a PP2A regulatory subunit, in the support of oncogenic phenotypes, including in vivo tumorigenicity/metastasis of pancreatic cancer cells. In this report, we have elucidated a novel role of PR55α-regulated PP2A in the activation of YAP oncoprotein, whose function is required for anchorage-independent growth during oncogenesis of solid tumors. Our data show two lines of YAP regulation by PR55α: (1) PR55α inhibits the MOB1-triggered autoactivation of LATS1/2 kinases, the core member of the Hippo pathway that inhibits YAP by inducing its proteasomal degradation and cytoplasmic retention and (2) PR55α directly interacts with and regulates YAP itself. Accordingly, PR55α is essential for YAP-promoted gene transcriptions, as well as for anchorage-independent growth, in which YAP plays a key role. In summary, current findings demonstrate a novel YAP activation mechanism based on the PR55α-regulated PP2A phosphatase.

Cigarette Smoke Induces Stem Cell Features of Pancreatic Cancer Cells via PAF1.

BACKGROUND & AIMS: Cigarette smoking is a major risk factor for pancreatic cancer. Aggressive pancreatic tumors contain cancer cells with stem cell features. We investigated whether cigarette smoke induces stem cell features in pancreatic cancer cells. METHODS: KrasG12D; Pdx1-Cre mice were exposed to cigarette smoke or clean air (controls) for up to 20 weeks; pancreata were collected and analyzed by histology, quantitative reverse transcription polymerase chain reaction, and confocal immunofluorescence microscopy. HPNE and Capan1 cells were exposed to cigarette smoke extract (CSE), nicotine and nicotine-derived carcinogens (NNN or NNK), or clean air (controls) for 80 days and evaluated for stem cell markers and features using flow cytometry-based autofluorescence, sphere formation, and immunoblot assays. Proteins were knocked down in cells with small interfering RNAs. We performed RNA sequencing analyses of CSE-exposed cells. We used chromatin immunoprecipitation assays to confirm the binding of FOS-like 1, AP-1 transcription factor subunit (FOSL1) to RNA polymerase II-associated factor (PAF1) promoter. We obtained pancreatic ductal adenocarcinoma (PDAC) and matched nontumor tissues (n = 15) and performed immunohistochemical analyses. RESULTS: Chronic exposure of HPNE and Capan1 cells to CSE caused them to increase markers of stem cells, including autofluorescence and sphere formation, compared with control cells. These cells increased expression of ABCG2, SOX9, and PAF1, via cholinergic receptor nicotinic alpha 7 subunit (CHRNA7) signaling to mitogen-activated protein kinase 1 and FOSL1. CSE-exposed pancreatic cells with knockdown of PAF1 did not show stem cell features. Exposure of cells to NNN and NNK led to increased expression of CHRNA7, FOSL1, and PAF1 along with stem cell features. Pancreata from KrasG12D; Pdx1-Cre mice exposed to cigarette smoke had increased levels of PAF1 mRNA and protein, compared with control mice, as well as increased expression of SOX9. Levels of PAF1 and FOSL1 were increased in PDAC tissues, especially those from smokers, compared with nontumor pancreatic tissue. CSE exposure increased expression of PHD-finger protein 5A, a pluripotent transcription factor and its interaction with PAF1. CONCLUSIONS: Exposure to cigarette smoke activates stem cell features of pancreatic cells, via CHRNA7 signaling and FOSL1 activation of PAF1 expression. Levels of PAF1 are increased in pancreatic tumors of humans and mice with chronic cigarette smoke exposure.

Inhibitors of telomerase and poly(ADP-ribose) polymerases synergize to limit the lifespan of pancreatic cancer cells.

Imetelstat (GRN163L) is a potent and selective inhibitor of telomerase. We have previously reported that GRN163L could shorten telomeres and limit the lifespan of CD18/HPAF and CAPAN1 pancreatic cancer cells. Here, we examined the effects of GRN163L on two other pancreatic cancer cell lines: AsPC1 and L3.6pl. In both lines, chronic exposure to GRN163L led to an initial shortening of telomeres followed by a stabilization of extremely short telomeres. In AsPC1 cells, telomere attrition eventually led to the induction of crisis and the loss of the treated population. In L3.6pl cells, crisis was transient and followed by the emergence of GRN163L-resistant cells, which could grow at increasing concentrations of GRN163L. The Shelterin complex is a telomere-associated complex that limits the access of telomerase to telomeres. The telomerase inhibitory function of this complex can be enhanced by drugs that block the poly(ADP-ribosyl)ation of its TRF1 and/or TRF2 subunits. Combined treatment of the GRN163L-resistant L3.6pl cells with GRN163L and 3-aminobenzamide (3AB), a general inhibitor of poly(ADP-ribose) polymerases, led to additional telomere shortening and limited the lifespan of the resistant cells. Results from this work suggest that inhibitors of telomerase and poly(ADP-ribose) polymerases can cooperate to limit the lifespan of pancreatic cancer cells.

Discovery and characterization of small molecule Rac1 inhibitors.

Aberrant activation of Rho GTPase Rac1 has been observed in various tumor types, including pancreatic cancer. Rac1 activates multiple signaling pathways that lead to uncontrolled proliferation, invasion and metastasis. Thus, inhibition of Rac1 activity is a viable therapeutic strategy for proliferative disorders such as cancer. Here we identified small molecule inhibitors that target the nucleotide-binding site of Rac1 through in silico screening. Follow up in vitro studies demonstrated that two compounds blocked active Rac1 from binding to its effector PAK1. Fluorescence polarization studies indicate that these compounds target the nucleotide-binding site of Rac1. In cells, both compounds blocked Rac1 binding to its effector PAK1 following EGF-induced Rac1 activation in a dose-dependent manner, while showing no inhibition of the closely related Cdc42 and RhoA activity. Furthermore, functional studies indicate that both compounds reduced cell proliferation and migration in a dose-dependent manner in multiple pancreatic cancer cell lines. Additionally, the two compounds suppressed the clonogenic survival of pancreatic cancer cells, while they had no effect on the survival of normal pancreatic ductal cells. These compounds do not share the core structure of the known Rac1 inhibitors and could serve as additional lead compounds to target pancreatic cancers with high Rac1 activity.

Biological determinants of radioresistance and their remediation in pancreatic cancer.

Despite recent advances in radiotherapy, a majority of patients diagnosed with pancreatic cancer (PC) do not achieve objective responses due to the existence of intrinsic and acquired radioresistance. Identification of molecular mechanisms that compromise the efficacy of radiation therapy and targeting these pathways is paramount for improving radiation response in PC patients. In this review, we have summarized molecular mechanisms associated with the radio-resistant phenotype of PC. Briefly, we discuss the reversible and irreversible biological consequences of radiotherapy, such as DNA damage and DNA repair, mechanisms of cancer cell survival and radiation-induced apoptosis following radiotherapy. We further describe various small molecule inhibitors and molecular targeting agents currently being tested in preclinical and clinical studies as potential radiosensitizers for PC. Notably, we draw attention towards the confounding effects of cancer stem cells, immune system, and the tumor microenvironment in the context of PC radioresistance and radiosensitization. Finally, we discuss the need for examining selective radioprotectors in light of the emerging evidence on radiation toxicity to non-target tissue associated with PC radiotherapy.

PR55α Subunit of Protein Phosphatase 2A Supports the Tumorigenic and Metastatic Potential of Pancreatic Cancer Cells by Sustaining Hyperactive Oncogenic Signaling.

The protein phosphatase 2 (PP2A) holoenzyme consists of a catalytic subunit, a scaffold subunit, and a regulatory subunit. Based on loss-of-function analysis using PP2A catalytic inhibitors or inhibition via tumor viral antigens, limited studies suggest that PP2A is a putative tumor suppressor. However, PP2A has also been shown to facilitate the activation of oncogenic signaling pathways when associated with specific regulatory subunits. In this study, we investigated the possible oncogenic role of PP2A in pancreatic cancer. We found a striking increase in the expression of PR55α (PPP2R2A), a PP2A regulatory subunit, in pancreatic cancer cells compared with normal pancreatic epithelial cells. Consistently, PR55α expression was markedly elevated in pancreatic ductal adenocarcinoma tissues compared with adjacent normal pancreatic tissues (P < 0.0001) and correlated with poor survival of pancreatic cancer patients (P < 0.0003). RNAi-mediated depletion of PR55α in pancreatic cancer cell lines resulted in diminished phosphorylation of both AKT and ERK1/2 (MAPK3/1) and decreased protein levels of ß-catenin (CTNNB1). Accordingly, pancreatic cancer cells with reduced PR55α expression exhibited significantly impaired properties of transformation, including attenuated cell growth, clonogenicity, mobility, and anchorage-independent growth. Moreover, orthotopic implantation of PR55α-depleted pancreatic cancer cells into nude mice resulted in markedly reduced tumorigenicity (P < 0.001) and distant metastases. Together, these results suggest that PR55α promotes pancreatic cancer development by sustaining hyperactivity of multiple oncogenic signaling pathways, including AKT, ERK, and Wnt. These studies also provide a basis for exploring PR55α as a diagnostic or therapeutic target in pancreatic cancer. Cancer Res; 76(8); 2243-53. ©2016 AACR.

CXCR2 signaling regulates KRAS(G¹²D)-induced autocrine growth of pancreatic cancer.

Pharmacological inhibition of RAS, the master regulator of pancreatic ductal adenocarcinoma (PDAC), continues to be a challenge. Mutations in various isoforms of RAS gene, including KRAS are known to upregulate CXC chemokines; however, their precise role in KRAS-driven pancreatic cancer remains unclear. In this report, we reveal a previously unidentified tumor cell-autonomous role of KRAS(G12D)-induced CXCR2 signaling in mediating growth of neoplastic PDAC cells. Progressively increasing expression of mCXCR2 and its ligands was detected in the malignant ductal cells of Pdx1-cre;LSL-Kras(G12D) mice. Knocking-down CXCR2 in KRAS(G12D)-bearing human pancreatic duct-derived cells demonstrated a significant decrease in the in vitro and in vivo tumor cell proliferation. Furthermore, CXCR2 antagonists showed selective growth inhibition of KRAS(G12D)-bearing cells in vitro. Intriguingly, both genetic and pharmacological inhibition of CXCR2 signaling in KRAS(G12D)-bearing pancreatic ductal cells reduced the levels of KRAS protein, strongly implying the presence of a KRAS-CXCR2 feed-forward loop. Together, these data demonstrate the role of CXCR2 signaling in KRAS(G12D)-induced growth transformation and progression in PDAC.

The OB-fold domain 1 of human POT1 recognizes both telomeric and non-telomeric DNA motifs.

The POT1 protein plays a critical role in telomere protection and telomerase regulation. POT1 binds single-stranded 5'-TTAGGGTTAG-3' and forms a dimer with the TPP1 protein. The dimer is recruited to telomeres, either directly or as part of the Shelterin complex. Human POT1 contains two Oligonucleotide/Oligosaccharide Binding (OB) fold domains, OB1 and OB2, which make physical contact with the DNA. OB1 recognizes 5'-TTAGGG whereas OB2 binds to the downstream TTAG-3'. Studies of POT1 proteins from other species have shown that some of these proteins are able to recognize a broader variety of DNA ligands than expected. To explore this possibility in humans, we have used SELEX to reexamine the sequence-specificity of the protein. Using human POT1 as a selection matrix, high-affinity DNA ligands were selected from a pool of randomized single-stranded oligonucleotides. After six successive rounds of selection, two classes of high-affinity targets were obtained. The first class was composed of oligonucleotides containing a cognate POT1 binding sites (5'-TTAGGGTTAG-3'). The second and more abundant class was made of molecules that carried a novel non-telomeric consensus: 5'-TNCANNAGKKKTTAGG-3' (where K = G/T and N = any base). Binding studies showed that these non-telomeric sites were made of an OB1-binding motif (TTAGG) and a non-telomeric motif (NT motif), with the two motifs recognized by distinct regions of the OB1 domain. POT1 interacted with these non-telomeric binding sites with high affinity and specificity, even when bound to its dimerization partner TPP1. This intrinsic ability of POT1 to recognize NT motifs raises the possibility that the protein may fulfill additional functions at certain non-telomeric locations of the genome, in perhaps gene transcription, replication, or repair.

Inhibition of RAC1 GTPase sensitizes pancreatic cancer cells to γ-irradiation.

Radiation therapy is a staple treatment for pancreatic cancer. However, owing to the intrinsic radioresistance of pancreatic cancer cells, radiation therapy often fails to increase survival of pancreatic cancer patients. Radiation impedes cancer cells by inducing DNA damage, which can activate cell cycle checkpoints. Normal cells possess both a G1 and G2 checkpoint. However, cancer cells are often defective in G1 checkpoint due to mutations/alterations in key regulators of this checkpoint. Accordingly, our results show that normal pancreatic ductal cells respond to ionizing radiation (IR) with activation of both checkpoints whereas pancreatic cancer cells respond to IR with G2/M arrest only. Overexpression/hyperactivation of Rac1 GTPase is detected in the majority of pancreatic cancers. Rac1 plays important roles in survival and Ras-mediated transformation. Here, we show that Rac1 also plays a critical role in the response of pancreatic cancer cells to IR. Inhibition of Rac1 using specific inhibitor and dominant negative Rac1 mutant not only abrogates IR-induced G2 checkpoint activation, but also increases radiosensitivity of pancreatic cancer cells through induction of apoptosis. These results implicate Rac1 signaling in the survival of pancreatic cancer cells following IR, raising the possibility that this pathway contributes to the intrinsic radioresistance of pancreatic cancer.

Radiation-induced signaling pathways that promote cancer cell survival (review).

Radiation therapy is a staple cancer treatment approach that has significantly improved local disease control and the overall survival of cancer patients. However, its efficacy is still limited by the development of radiation resistance and the presence of residual disease after therapy that leads to cancer recurrence. Radiation impedes cancer cell growth by inducing cytotoxicity, mainly caused by DNA damage. However, radiation can also simultaneously induce multiple pro-survival signaling pathways, such as those mediated by AKT, ERK and ATM/ATR, which can lead to suppression of apoptosis, induction of cell cycle arrest and/or initiation of DNA repair. These signaling pathways act conjointly to reduce the magnitude of radiation-induced cytotoxicity and promote the development of radioresistance in cancer cells. Thus, targeting these pro-survival pathways has great potential for the radiosensitization of cancer cells. In the present review, we summarize the current literature on how these radiation­activated signaling pathways promote cancer cell survival.

Context-dependent function of the deubiquitinating enzyme USP9X in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and deadly malignancies. Recently, the deubiquitinating protease USP9X has been shown to behave as an oncogene in a number of neoplasms, including those of breast, brain, colon, esophagus and lung, as well as KRAS wild-type PDAC. However, other studies suggest that USP9X may function as a tumor-suppressor in a murine PDAC model when USP9X expression is depleted during early pancreatic development. To address the conflicting findings surrounding the role of USP9X in PDAC, we examined the effects of knocking down USP9X in five human PDAC cell lines (BxPC3, Capan1, CD18, Hs766T, and S2-013). We demonstrate that knocking down USP9X in each of the PDAC cell lines reduces their anchorage-dependent growth. Using an inducible shRNA system to knock down USP9X in both BxPC3 and Capan1 cells, we also determined that USP9X is necessary for the anchorage-independent growth. In addition, knockdown of USP9X alters the cell cycle profile of BxPC3 cells and increases their invasive capacity. Finally, we show that an inhibitor of deubiquitinating proteases, WP1130, induces significant cytotoxicity in each of the five PDAC cell lines tested. Overall, our work and the work of others indicate that the function and role of USP9X is highly context-dependent. Although USP9X may function as a tumor-suppressor during the establishment of PDAC, data presented here argue that USP9X promotes cell growth in advanced PDAC cells when PDAC is typically diagnosed. Hence, USP9X may be a promising therapeutic target for the treatment of advanced PDAC.