RNA Polymerase II-Associated Factor 1 Regulates Stem Cell Features of Pancreatic Cancer Cells, Independently of the PAF1 Complex, via Interactions With PHF5A and DDX3.

BACKGROUND & AIMS: It is not clear how pancreatic cancer stem cells (CSCs) are regulated, resulting in ineffective treatments for pancreatic cancer. PAF1, a RNA polymerase II-associated factor 1 complex (PAF1C) component, maintains pluripotency of stem cells, by unclear mechanisms, and is a marker of CSCs. We investigated mechanisms by which PAF1 maintains CSCs and contributes to development of pancreatic tumors. METHODS: Pancreatic cancer cell lines were engineered to knockdown PAF1 using inducible small hairpin RNAs. These cells were grown as orthotopic tumors in athymic nude mice and PAF1 knockdown was induced by administration of doxycycline in drinking water. Tumor growth and metastasis were monitored via IVIS imaging. CSCs were isolated from pancreatic cancer cell populations using flow cytometry and characterized by tumor sphere formation, tumor formation in nude mice, and expression of CSC markers. Isolated CSCs were depleted of PAF1 using the CRISPR/Cas9 system. PAF1-regulated genes in CSCs were identified via RNA-seq and PCR array analyses of cells with PAF1 knockdown. Proteins that interact with PAF1 in CSCs were identified by immunoprecipitations and mass spectrometry. We performed chromatin immunoprecipitation sequencing of CSCs to confirm the binding of the PAF1 sub-complex to target genes. RESULTS: Pancreatic cancer cells depleted of PAF1 formed smaller and fewer tumor spheres in culture and orthotopic tumors and metastases in mice. Isolated CSCs depleted of PAF1 downregulated markers of self-renewal (NANOG, SOX9, and ß-CATENIN), of CSCs (CD44v6, and ALDH1), and the metastasis-associated gene signature, compared to CSCs without knockdown of PAF1. The role of PAF1 in CSC maintenance was independent of its RNA polymerase II-associated factor 1 complex component identity. We identified DDX3 and PHF5A as proteins that interact with PAF1 in CSCs and demonstrated that the PAF1-PHF5A-DDX3 sub-complex bound to the promoter region of Nanog, whose product regulates genes that control stemness. Levels of the PAF1-DDX3 and PAF1-PHF5A were increased and co-localized in human pancreatic tumor specimens, human pancreatic tumor-derived organoids, and organoids derived from tumors of KPC mice, compared with controls. Binding of DDX3 and PAF1 to the Nanog promoter, and the self-renewal capacity of CSCs, were decreased in cells incubated with the DDX3 inhibitor RK-33. CSCs depleted of PAF1 downregulated genes that regulate stem cell features (Flot2, Taz, Epcam, Erbb2, Foxp1, Abcc5, Ddr1, Muc1, Pecam1, Notch3, Aldh1a3, Foxa2, Plat, and Lif). CONCLUSIONS: In pancreatic CSCs, PAF1 interacts with DDX3 and PHF5A to regulate expression of NANOG and other genes that regulate stemness. Knockdown of PAF1 reduces the ability of orthotopic pancreatic tumors to develop and progress in mice and their numbers of CSCs. Strategies to target the PAF1-PHF5A-DDX3 complex might be developed to slow or inhibit progression of pancreatic cancer.

PKCε inhibits isolation and stemness of side population cells via the suppression of ABCB1 transporter and PI3K/Akt, MAPK/ERK signaling in renal cell carcinoma cell line 769P.

Protein kinase C epsilon (PKCε), a member of the novel PKC family, is known to be a transforming oncogene and tumor biomarker for many human solid cancers including renal cell carcinoma (RCC). We isolated side population (SP) cells from the RCC 769P cell line, and proved that those cells possess cancer stem cell (CSC) characteristics. In this study, to identify the function of PKCε in cancer stemness of 769P SP cells, we reduced the expression of PKCε in those cells, following the results demonstrated that PKCε depletion had a negative correlation with the existence of SP cells in 769P cell line. Down-regulation of PKCε also suppresses the CSC potential of sorted 769P SP cells in several ways: proliferation potential, resistance to chemotherapeutics and in vivo tumor formation ability. Our study also reveals that PKCε is associated with ABCB1 and this association probably contributed to the SP cells isolation from 769P cell line. Furthermore, the expression of ABCB1 is directly regulated by PKCε. Additionally, after the depletion of PKCε, the phosphorylation of pAkt, pStat3 and pERK was apparently suppressed in 769P SP cells, whereas PKCε overexpression could promote the phosphorylation of AKT, STAT3 and ERK in 769P Non-SP cells. Overall, PKCε down-regulation suppresses sorting and the cancer stem-like phenotype of RCC 769P SP cells through the regulation of ABCB1 transporter and the PI3K/Akt, Stat3 and MAPK/ERK pathways that are dependent on the phosphorylation effects. Thus, PKCε may work as an important mediator in cancer stem cell pathogenesis of renal cell cancer.

Urotensin II inhibited the proliferation of cardiac side population cells in mice during pressure overload by JNK-LRP6 signalling.

Cardiac side population cells (CSPs) are promising cell resource for the regeneration in diseased heart as intrinsic cardiac stem cells. However, the relative low ratio of CSPs in the heart limited the ability of CSPs to repair heart and improve cardiac function effectively under pathophysiological condition. Which factors limiting the proliferation of CSPs in diseased heart are unclear. Here, we show that urotensin II (UII) regulates the proliferation of CSPs by c-Jun N-terminal kinase (JNK) and low density lipoprotein receptor-related protein 6 (LRP6) signalling during pressure overload. Pressure overload greatly upregulated UII level in plasma, UII receptor (UT) antagonist, urantide, promoted CSPs proliferation and improved cardiac dysfunction during chronic pressure overload. In cultured CSPs subjected to mechanical stretch (MS), UII significantly inhibited the proliferation by UT. Nanofluidic proteomic immunoassay showed that it is the JNK activation, but not the extracellular signal-regulated kinase signalling, that involved in the UII-inhibited- proliferation of CSPs during pressure overload. Further analysis in vitro indicated UII-induced-phospho-JNK regulates phosphorylation of LRP6 in cultured CSPs after MS, which is important in the inhibitory effect of UII on the CSPs during pressure overload. In conclusion, UII inhibited the proliferation of CSPs by JNK/LRP6 signalling during pressure overload. Pharmacological inhibition of UII promotes CSPs proliferation in mice, offering a possible therapeutic approach for cardiac failure induced by pressure overload.

STAT3 & Cytochrome P450 2C9: a novel signaling pathway in liver cancer stem cells.

Although cancer stem cells (CSCs) are believed to be the key drivers in tumor growth and resistance to therapy, the specific signaling of CSCs is largely unknown. In this study, we evaluated the roles of hypoxia and STAT3 signaling on the treatment resistance of CSCs. Side population (SP) cell analysis and sorting were used to detect subpopulations that function as CSCs. Huh-7 cells, doxorubicin, sulfaphenazole (a CYP2C9 inhibitor), and AG490 (a STAT3 inhibitor) were used in this study. Cell growth and apoptosis were assessed using MTS assays, and apoptotic and kinase signaling pathways were explored by immunoblotting. Treatment with IL-6 induced STAT3 activation more significantly in SP than non-SP cells. Hypoxia induced SP cell proliferation, and microarray analysis showed that the expression of CYP2C9 was significantly increased in hypoxic than normoxic SP cells. Although hypoxic SP cells were less sensitive to doxorubicin-induced apoptosis, pretreatment with sulfaphenazole sensitized hypoxic SP cells to doxorubicin cytotoxicity. These results indicate that STAT3 is critical for CSC survival and hypoxia-inducible CYP2C9 expression is responsible the doxorubicin resistance of CSCs under hypoxic conditions. Thus, the selective inhibition of CYP2C9 and STAT3 may be implicated in the sensitization of CSCs to anti-cancer treatment, particularly in advanced cases.

Rac1 targeting suppresses human non-small cell lung adenocarcinoma cancer stem cell activity.

The cancer stem cell (CSC) theory predicts that a small fraction of cancer cells possess unique self-renewal activity and mediate tumor initiation and propagation. However, the molecular mechanisms involved in CSC regulation remains unclear, impinging on effective targeting of CSCs in cancer therapy. Here we have investigated the hypothesis that Rac1, a Rho GTPase implicated in cancer cell proliferation and invasion, is critical for tumor initiation and metastasis of human non-small cell lung adenocarcinoma (NSCLA). Rac1 knockdown by shRNA suppressed the tumorigenic activities of human NSCLA cell lines and primary patient NSCLA specimens, including effects on invasion, proliferation, anchorage-independent growth, sphere formation and lung colonization. Isolated side population (SP) cells representing putative CSCs from human NSCLA cells contained elevated levels of Rac1-GTP, enhanced in vitro migration, invasion, increased in vivo tumor initiating and lung colonizing activities in xenografted mice. However, CSC activity was also detected within the non-SP population, suggesting the importance of therapeutic targeting of all cells within a tumor. Further, pharmacological or shRNA targeting of Rac1 inhibited the tumorigenic activities of both SP and non-SP NSCLA cells. These studies indicate that Rac1 represents a useful target in NSCLA, and its blockade may have therapeutic value in suppressing CSC proliferation and metastasis.

Preclinical testing of the Akt inhibitor triciribine in T-cell acute lymphoblastic leukemia.

Over the past 20 years, survival rates of T-cell acute lymphoblastic leukemia (T-ALL) patients have improved, mainly because of advances in polychemotherapy protocols. Despite these improvements, we still need novel and less toxic treatment strategies targeting aberrantly activated signaling networks which increase proliferation, survival, and drug resistance of T-ALL cells. One such network is represented by the phosphatidylinositol 3-kinase (PI3K)/Akt axis. PI3K inhibitors have displayed some promising effects in preclinical models of T-ALL. Here, we have analyzed the therapeutic potential of the Akt inhibitor, triciribine, in T-ALL cell lines. Triciribine caused cell cycle arrest and caspase-dependent apoptosis. Western blots demonstrated a dose-dependent dephosphorylation of Akt1/Akt2, and of mammalian target of rapamycin complex 1 downstream targets in response to triciribine. Triciribine induced autophagy, which could be interpreted as a defensive mechanism, because an autophagy inhibitor (chloroquine) increased triciribine-induced apoptosis. Triciribine synergized with vincristine, a chemotherapeutic drug employed for treating T-ALL patients, and targeted the side population of T-ALL cell lines, which might correspond to leukemia initiating cells. Our findings indicate that Akt inhibition, either alone or in combination with chemotherapeutic drugs, may serve as an efficient treatment towards T-ALL cells requiring upregulation of this signaling pathway for their proliferation and survival.

Human adipose-derived side population stem cells cultured on demineralized bone matrix for bone tissue engineering.

BACKGROUND: Tissue engineering of new bone relies on the combination and application of osteoconductive, osteoinductive, and osteogenic elements. Natural scaffolds, such as demineralized bone matrix (DBM), contain collagenous networks with growth factors such as bone morphogenetic protein-2. Stem cells from readily available sources, including discarded adipose tissue, have the propensity to differentiate into bone. The present study examines a multi-component technique consisting of a novel side population of adipose stem cells cultured on DBM for tissue engineering applications. METHODS: Adipose-derived side population stem cells were cultured on DBM for up to 14 days. Cell proliferation, alkaline phosphatase activity, extracellular matrix protein production, and calcium-containing mineral deposit formation were assayed. Ectopic bone formation in a rat model was also evaluated. RESULTS: Side population stem cells attached to and proliferated on DBM while generating markers of new bone formation. When these cell/substrate composites were implanted into an ectopic model, newly formed bone was 30% greater than that of DBM alone. CONCLUSIONS: Novel populations of adipose-derived stem cells cultured on DBM compose a system that develops new bone matrix in vitro and in vivo. This strategy provides a novel approach using naturally occurring materials for bone repair in tissue engineering applications.