RUNX3 Controls a Metastatic Switch in Pancreatic Ductal Adenocarcinoma.

For the majority of patients with pancreas cancer, the high metastatic proclivity is life limiting. Some patients, however, present with and succumb to locally destructive disease. A molecular understanding of these distinct disease manifestations can critically inform patient management. Using genetically engineered mouse models, we show that heterozygous mutation of Dpc4/Smad4 attenuates the metastatic potential of Kras(G12D/+);Trp53(R172H/+) pancreatic ductal adenocarcinomas while increasing their proliferation. Subsequent loss of heterozygosity of Dpc4 restores metastatic competency while further unleashing proliferation, creating a highly lethal combination. Expression levels of Runx3 respond to and combine with Dpc4 status to coordinately regulate the balance between cancer cell division and dissemination. Thus, Runx3 serves as both a tumor suppressor and promoter in slowing proliferation while orchestrating a metastatic program to stimulate cell migration, invasion, and secretion of proteins that favor distant colonization. These findings suggest a model to anticipate likely disease behaviors in patients and tailor treatment strategies accordingly.

Targeted depletion of an MDSC subset unmasks pancreatic ductal adenocarcinoma to adaptive immunity.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDA) is characterised by a robust desmoplasia, including the notable accumulation of immunosuppressive cells that shield neoplastic cells from immune detection. Immune evasion may be further enhanced if the malignant cells fail to express high levels of antigens that are sufficiently immunogenic to engender an effector T cell response. OBJECTIVE: To investigate the predominant subsets of immunosuppressive cancer-conditioned myeloid cells that chronicle and shape the progression of pancreas cancer. We show that selective depletion of one subset of myeloid-derived suppressor cells (MDSC) in an autochthonous, genetically engineered mouse model (GEMM) of PDA unmasks the ability of the adaptive immune response to engage and target tumour epithelial cells. METHODS: A combination of in vivo and in vitro studies were performed employing a GEMM that faithfully recapitulates the cardinal features of human PDA. The predominant cancer-conditioned myeloid cell subpopulation was specifically targeted in vivo and the biological outcomes determined. RESULTS: PDA orchestrates the induction of distinct subsets of cancer-associated myeloid cells through the production of factors known to influence myelopoiesis. These immature myeloid cells inhibit the proliferation and induce apoptosis of activated T cells. Targeted depletion of granulocytic MDSC (Gr-MDSC) in autochthonous PDA increases the intratumoral accumulation of activated CD8 T cells and apoptosis of tumour epithelial cells and also remodels the tumour stroma. CONCLUSIONS: Neoplastic ductal cells of the pancreas induce distinct myeloid cell subsets that promote tumour cell survival and accumulation. Targeted depletion of a single myeloid subset, the Gr-MDSC, can unmask an endogenous T cell response, disclosing an unexpected latent immunity and invoking targeting of Gr-MDSC as a potential strategy to exploit for treating this highly lethal disease.

Inhibition of Hedgehog signaling enhances delivery of chemotherapy in a mouse model of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) is among the most lethal human cancers in part because it is insensitive to many chemotherapeutic drugs. Studying a mouse model of PDA that is refractory to the clinically used drug gemcitabine, we found that the tumors in this model were poorly perfused and poorly vascularized, properties that are shared with human PDA. We tested whether the delivery and efficacy of gemcitabine in the mice could be improved by coadministration of IPI-926, a drug that depletes tumor-associated stromal tissue by inhibition of the Hedgehog cellular signaling pathway. The combination therapy produced a transient increase in intratumoral vascular density and intratumoral concentration of gemcitabine, leading to transient stabilization of disease. Thus, inefficient drug delivery may be an important contributor to chemoresistance in pancreatic cancer.

Targets, trials, and travails in pancreas cancer.

Pancreas cancer is a highly aggressive and rapidly fatal disease. The current standard of care for advanced disease improves survival modestly at best and provides palliation for a minority of patients. The need for new therapies is undisputed. This article describes new therapeutic strategies currently under investigation and discusses possible reasons that others have failed. New potential targets in the treatment of this formidable disease are suggested based on recent findings.

Kras(G12D) and Smad4/Dpc4 haploinsufficiency cooperate to induce mucinous cystic neoplasms and invasive adenocarcinoma of the pancreas.

Oncogenic Kras initiates pancreatic tumorigenesis, while subsequent genetic events shape the resultant disease. We show here that concomitant expression of Kras(G12D) and haploinsufficiency of the Smad4/Dpc4 tumor suppressor gene engenders a distinct class of pancreatic tumors, mucinous cystic neoplasms (MCNs), which culminate in invasive ductal adenocarcinomas. Disease evolves along a progression scheme analogous to, but distinct from, the classical PanIN-to-ductal adenocarcinoma sequence, and also portends a markedly different prognosis. Progression of MCNs is accompanied by LOH of Dpc4 and mutation of either p53 or p16. Thus, these distinct phenotypic routes to invasive adenocarcinoma nevertheless share the same overall mutational spectra. Our findings suggest that the sequence, as well as the context, in which these critical mutations are acquired helps determine the ensuing pathology.

Proteasome inhibitors sensitize colon carcinoma cells to TRAIL-induced apoptosis via enhanced release of Smac/DIABLO from the mitochondria.

The synergistic interaction between proteasome inhibitors and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a promising approach to induce cell death in tumor cells. However, the molecular and biochemical mechanisms of this synergism have been proven to be cell type specific. We therefore focused our investigation on TRAIL-resistant colon carcinoma cells in this study. DNA fragmentation, mitochondrial membrane depolarization and increased caspase-3-like enzyme activity was exclusively induced only by combined treatment with proteasome inhibitors (epoxomicin, MG132, bortezomib/PS-341) and TRAIL. The expression level of anti-apoptotic proteins (XIAP, survivin, Bcl-2, Bcl-XL), regulated by NF-kappaB transcription factor, was not effected by any of these treatments. TRAIL alone induced only partial activation of caspase-3 (p20), while the combination of TRAIL and proteasome inhibition led to the full proteolytic activation of caspase-3 (p17). Only the combination treatment induced marked membrane depolarization and the release of cytochrome c, HtrA2/Omi and Smac/DIABLO. Apoptosis-inducing factor (AIF) was not released in any of these conditions. These results are consistent with a model where the full activation of caspase-3 by caspase-8 is dependent on the release of Smac/DIABLO in response to the combined treatment. This molecular mechanism, independent of the inhibition NF-kappaB activity, may provide rationale for the combination treatment of colon carcinomas with proteasome inhibitors and recombinant TRAIL or agonistic antibody of TRAIL receptors.

FOXO1a acts as a selective tumor suppressor in alveolar rhabdomyosarcoma.

Rhabdomyosarcoma (RMS), the most common pediatric soft-tissue sarcoma, has two major histological subtypes: embryonal RMS (ERMS), which has a favorable prognosis, and alveolar RMS (ARMS), which has a poor outcome. Although both forms of RMS express muscle cell-specific markers, only ARMS cells express PAX3-FOXO1a or PAX7-FOXO1a chimeric proteins. In mice, Pax3 and Pax7 play key roles in muscle cell development and differentiation, and FoxO1a regulates myoblast differentiation and fusion; thus, the aberrant regulation of these proteins may contribute to the development of ARMS. In this paper, we report that FOXO1a is not expressed in primary ARMS tumors or ARMS-derived tumor cell lines and that restoration of FOXO1a expression in ARMS cells is sufficient to induce cell cycle arrest and apoptosis. Strikingly, the effects of FOXO1a are selective, as enforced expression of FOXO1a in ERMS-derived tumor cell lines had no effect. Furthermore, FOXO1a induced apoptosis in ARMS by directly activating the transcription of caspase-3. We conclude that FOXO1a is a potent and specific tumor suppressor in ARMS, suggesting that agents that restore or augment FOXO1a activity may be effective as ARMS therapeutics.

Reactive oxygen species regulate caspase activation in tumor necrosis factor-related apoptosis-inducing ligand-resistant human colon carcinoma cell lines.

The effects of reactive oxygen species (ROS) on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in solid cancers have yet to be clearly defined. In this study, we found that the classic uncoupler of oxidative phosphorylation, carbonyl cyanide m-chlorophenylhydrazone (CCCP), induced a reduction in DeltaPsim and generation of ROS. This uncoupling effect enhanced TRAIL-induced apoptosis in TRAIL-resistant human colon carcinoma cell lines (RKO, HT29, and HCT8). Sensitization was inhibited by benzyloxycarbonyl-valine-alanine-aspartate fluoromethylketone, indicating the requirement for caspase activation. CCCP per se did not induce apoptosis or release of proapoptotic factors from mitochondria. Generation of ROS by CCCP was responsible for TRAIL-induced Bax and caspase activation because scavenging ROS completely abrogated apical caspase-8 activation and further downstream events leading to cell death. Overexpression of Bcl-2 did not prevent the initial loss of DeltaPsim and ROS generation following CCCP treatment, but did prevent cell death following TRAIL and CCCP exposure. Uncoupling of mitochondria also facilitated TRAIL-induced release of proapoptotic factors. X-linked inhibitor of apoptosis overexpression abrogated TRAIL-induced apoptosis in the presence of CCCP and decreased initiator procaspase-8 processing, indicating that additional processing of caspase-8 required initiation of a mitochondrial amplification loop via effector caspases. Of interest, depletion of caspase-9 in RKO cells did not protect cells from TRAIL/CCCP-induced apoptosis, indicating that apoptosis occurred via a caspase-9-independent pathway. Data suggest that in the presence of mitochondrial-derived ROS, TRAIL induced mitochondrial release of Smac/DIABLO and inactivation of X-linked inhibitor of apoptosis through caspase-9-independent activation of caspase 3.

Casein kinase II (CK2) enhances death-inducing signaling complex (DISC) activity in TRAIL-induced apoptosis in human colon carcinoma cell lines.

Protein kinase casein kinase II (CK2) is increased in response to diverse growth stimuli, as well as being elevated in many human cancers examined. We have demonstrated that CK2 is a key survival factor that protects human colon carcinoma cells from TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. We determined that inhibition of CK2 phosphorylation events by DRB (5,6-dichlorobenzimidazole) resulted in dramatic sensitization of tumor cells to TRAIL-induced apoptosis, in the absence of effects in normal cells. Sensitization was caspase dependent, and independent of regulation via NF-kappaB. Further, inhibition of phosphorylation by CK2 did not modify the expression level of antiapoptotic proteins. Analysis of TRAIL-induced death-inducing signaling complex (DISC) formation demonstrated enhanced formation of the DISC, enhanced cleavage of caspase-8 and cleavage of Bid in the presence of DRB, thereby facilitating the release of proapoptotic factors from the mitochondria with subsequent downregulation of the expression of XIAP and c-IAP1. Further, silencing of CK2alpha in HT29 cells following transfection of CK2alpha shRNA abrogated CK2 kinase activity while simultaneously increasing TRAIL sensitivity. These findings demonstrate that CK2 plays a critical antiapoptotic role by conferring resistance to TRAIL at the level of the DISC.

Casein kinase I attenuates tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by regulating the recruitment of fas-associated death domain and procaspase-8 to the death-inducing signaling complex.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a wide variety of malignant cell lines, in contrast to normal cells, but with considerable heterogeneity in response. Death receptor-mediated apoptosis may be attenuated by a variety of different mechanisms, including phosphorylation-based signaling pathways. We have demonstrated that casein kinase I can attenuate TRAIL-induced apoptosis in human cell lines derived from colon adenocarcinoma (HT29 and HCT8) and pediatric rhabdomyosarcoma (JR1). Inhibition of casein kinase I (CKI) phosphorylation events in HT29, HCT8, and JR1 cells by CKI-7 dramatically increased apoptosis after exposure to TRAIL, in the absence of apoptosis induced by TRAIL treatment alone. CKI inhibition enhanced the recruitment of Fas-associated death domain and procaspase-8 to the death-inducing signaling complex after TRAIL treatment and enhanced cleavage of procaspase-8 at the death-inducing signaling complex. In HT29 cells studied further, rapid cleavage of caspase-8, caspase-3, Bid, and the caspase substrate poly(ADP-ribose) polymerase occurred when CKI-7 and TRAIL were combined. Overexpression of Bcl-2, Bcl-xL, or mutant DN-Fas-associated death domain protected HT29 cells from TRAIL-induced apoptosis in the presence of the CKI inhibitor. In addition, TRAIL combined with CKI-7 promoted the release of cytochrome c, Smac/DIABLO, HtrA2/Omi, and AIF from the mitochondria and down-regulated the expression of XIAP and c-IAP1. Small hairpin RNAs directed against CKI revealed that the CKIalpha isoform contributed significantly to the inhibition of TRAIL-induced apoptosis. These findings suggest that CKIalpha plays an antiapoptotic role through the generation of phosphorylated sites at the level of the death-inducing signaling complex, thereby conferring resistance to caspase cleavage mediated by TRAIL.

Influence of casein kinase II in tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human rhabdomyosarcoma cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis via the death receptors DR4 and DR5 in transformed cells in vitro and exhibits potent antitumor activity in vivo with minor side effects. Protein kinase casein kinase II (CK2) is increased in response to diverse growth stimuli and is aberrantly elevated in a variety of human cancers. Rhabdomyosarcoma tumors are the most common soft-tissue sarcoma in childhood. In this investigation, we demonstrate that CK2 is a key survival factor that protects tumor cells from TRAIL-induced apoptosis. We have demonstrated that inhibition of CK2 phosphorylation events by 5,6-dichlorobenzimidazole (DRB) resulted in dramatic sensitization of tumor cells to TRAIL-induced apoptosis. CK2 inhibition also induced rapid cleavage of caspase-8, -9, and -3, as well as the caspase substrate poly(ADP-ribose) polymerase after TRAIL treatment. Overexpression of Bcl-2 protected cells from TRAIL-induced apoptosis in the presence of the CK2 inhibitor. Death signaling by TRAIL in these cells was Fas-associated death domain and caspase dependent because dominant negative Fas-associated death domain or the cowpox interleukin 1beta-converting enzyme inhibitor protein cytokine response modifier A prevented apoptosis in the presence of DRB. Analysis of death-inducing signaling complex (DISC) formation demonstrated that inhibition of CK2 by DRB increased the level of recruitment of procaspase-8 to the DISC and enhanced caspase-8-mediated cleavage of Bid, thereby increasing the release of the proapoptotic factors cytochrome c, HtrA2/Omi, Smac/DIABLO, and apoptosis inducing factor (AIF) from the mitochondria, with subsequent degradation of X-linked inhibitor of apoptosis protein (XIAP). To further interfere with CK2 function, JR1 and Rh30 cells were transfected with either short hairpin RNA targeted to CK2alpha or kinase-inactive CK2alpha (K68M) or CK2alpha' (K69M). Data show that the CK2 kinase activity was abrogated and that TRAIL sensitivity in both cell lines was increased. Silencing of CK2alpha expression with short hairpin RNA was also associated with degradation of XIAP. These findings suggest that CK2 regulates TRAIL signaling in rhabdomyosarcoma by modulating TRAIL-induced DISC formation and XIAP expression.

Rottlerin sensitizes colon carcinoma cells to tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis via uncoupling of the mitochondria independent of protein kinase C.

Signaling pathways involved in survival responses may attenuate the apoptotic response to the cytotoxic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in human colon carcinomas. In six lines examined, three were sensitive (GC(3)/c1, VRC(5)/c1, HCT116), HT29 demonstrated intermediate sensitivity, and RKO and HCT8 were resistant to TRAIL-induced apoptosis. Calphostin c [an inhibitor of classic and novel isoforms of protein kinase C (PKC)] sensitized five of six cell lines to TRAIL, whereas Go6976, (inhibitor of classic PKC isoforms), did not influence TRAIL sensitivity. Rottlerin, an inhibitor of novel isoforms of PKC, specifically PKC delta, sensitized five of six cell lines to TRAIL-induced apoptosis, suggesting that PKC delta may be involved in the mechanism of TRAIL resistance. Transfection of HCT116 with a proapoptotic cleaved fragment of PKC delta or an antiapoptotic full-length PKC delta did not influence the sensitivity of HCT116 to TRAIL. Furthermore, the incubation of HCT116 or RKO with phorbol myristate acetate for 16 h, which down-regulated the expression of novel PKC isoforms, also did not influence sensitivity to TRAIL either in the absence or presence of rottlerin. However, after 15-min incubation with rottlerin, mitochondrial membrane potential (Delta psi m) was dramatically reduced in RKO cells, and, in cells subsequently treated with TRAIL, rapid apoptosis was evident within 8 h. Calphostin c, but not Go6976, also caused a decrease in Delta psi m. In RKO, rottlerin induced the release of cytochrome c, HtrA2/Omi, Smac/DIABLO, and AIF from the mitochondria, potentiated in combination with TRAIL, with concomitant caspase activation and down-regulation of XIAP. In HT29, the release of proapoptotic factors was demonstrated only when rottlerin and TRAIL were combined, and Bcl-2 overexpression inhibited this release and the induction of apoptosis. TRAIL-induced apoptosis was not influenced by rottlerin or Bcl-2 overexpression in type I (GC(3)/c1) cells. Data suggest that rottlerin affects mitochondrial function independent of PKC delta, thereby sensitizing cells to TRAIL, and that mitochondria constitute an important target in overcoming inherent resistance to TRAIL in colon carcinomas.