Oncogenic roles of PRL-3 in FLT3-ITD induced acute myeloid leukaemia

FLT3-ITD mutations are prevalent mutations in acute myeloid leukaemia (AML). PRL-3, a metastasis-associated phosphatase, is a downstream target of FLT3-ITD. This study investigates the regulation and function of PRL-3 in leukaemia cell lines and AML patients associated with FLT3-ITD mutations. PRL-3 expression is upregulated by the FLT3-STAT5 signalling pathway in leukaemia cells, leading an activation of AP-1 transcription factors via ERK and JNK pathways. PRL-3-depleted AML cells showed a significant decrease in cell growth. Clinically, high PRL-3 mRNA expression was associated with FLT3-ITD mutations in four independent AML datasets with 1158 patients. Multivariable Cox-regression analysis on our Cohort 1 with 221 patients identified PRL-3 as a novel prognostic marker independent of other clinical parameters. Kaplan-Meier analysis showed high PRL-3 mRNA expression was significantly associated with poorer survival among 491 patients with normal karyotype. Targeting PRL-3 reversed the oncogenic effects in FLT3-ITD AML models in vitro and in vivo. Herein, we suggest that PRL-3 could serve as a prognostic marker to predict poorer survival and as a promising novel therapeutic target for AML patients.

Th1 not Th17 cells drive spontaneous MS-like disease despite a functional regulatory T cell response

Multiple sclerosis is considered a disease of complex autoimmune etiology, yet there remains a lack of consensus as to specific immune effector mechanisms. Recent analyses of experimental autoimmune encephalomyelitis, the common mouse model of multiple sclerosis, have investigated the relative contribution of Th1 and Th17 CD4 T cell subsets to initial autoimmune central nervous system (CNS) damage. However, inherent in these studies are biases influenced by the adjuvant and toxin needed to break self-tolerance. We investigated spontaneous CNS disease in a clinically relevant, humanized, T cell receptor transgenic mouse model. Mice develop spontaneous, ascending paralysis, allowing unbiased characterization of T cell immunity in an HLA-DR15-restricted T cell repertoire. Analysis of naturally progressing disease shows that IFN?(+) cells dominate disease initiation with IL-17(+) cells apparent in affected tissue only once disease is established. Tregs accumulate in the CNS but are ultimately ineffective at halting disease progression. However, ablation of Tregs causes profound acceleration of disease, with uncontrolled infiltration of lymphocytes into the CNS. This synchronous, severe disease allows characterization of the responses that are deregulated in exacerbated disease: the correlation is with increased CNS CD4 and CD8 IFN? responses. Recovery of the ablated Treg population halts ongoing disease progression and Tregs extracted from the central nervous system at peak disease are functionally competent to regulate myelin specific T cell responses. Thus, in a clinically relevant mouse model of MS, initial disease is IFN? driven and the enhanced central nervous system responses unleashed through Treg ablation comprise IFN? cytokine production by CD4 and CD8 cells, but not IL-17 responses.

Dose, dose-rate and field size effects on cell survival following exposure to non-uniform radiation fields

For the delivery of intensity-modulated radiation therapy (IMRT), highly modulated fields are used to achieve dose conformity across a target tumour volume. Recent in vitro evidence has demonstrated significant alterations in cell survival occurring out-of-field which cannot be accounted for on the basis of scattered dose. The radiobiological effect of area, dose and dose-rate on out-of-field cell survival responses following exposure to intensity-modulated radiation fields is presented in this study. Cell survival was determined by clonogenic assay in human prostate cancer (DU-145) and primary fibroblast (AG0-1522) cells following exposure to different modulated field configurations delivered using a X-Rad 225 kVp x-ray source. Uniform survival responses were compared to in- and out-of-field responses in which 25-99% of the cell population was shielded. Dose delivered to the out-of-field region was varied from 1.6-37.2% of that delivered to the in-field region using different levels of brass shielding. Dose rate effects were determined for 0.2-4 Gy min⁻¹ for uniform and modulated exposures with no effect seen in- or out-of-field. Survival responses showed little dependence on dose rate and area in- and out-of-field with a trend towards increased survival with decreased in-field area. Out-of-field survival responses were shown to scale in proportion to dose delivered to the in-field region and also local dose delivered out-of-field. Mathematical modelling of these findings has shown survival response to be highly dependent on dose delivered in- and out-of-field but not on area or dose rate. These data provide further insight into the radiobiological parameters impacting on cell survival following exposure to modulated irradiation fields highlighting the need for refinement of existing radiobiological models to incorporate non-targeted effects and modulated dose distributions.

Induction of signalling in non-erythroid cells by pharmacological levels of erythropoietin

Erythropoiesis is maintained by the hormone erythropoietin (Epo) binding to its cognate receptor (EpoR) on erythroid progenitor cells. The Epo-EpoR interaction initiates a signal transduction process that regulates the survival, growth and differentiation of these cells. Originally perceived as highly lineage-restricted, Epo is now recognised to have pleiotropic effects extending beyond the maintenance of red cell mass. Functional interactions between Epo and EpoR have been demonstrated in numerous cells and tissues. EpoR expression on neoplastic cells leads to concern that recombinant human erythropoietin, used to treat anaemia in cancer patients, may augment tumour growth. Here we demonstrate that EPO, at pharmacological concentrations, can activate three major signalling cascades, viz. the Jak2/STAT5, Ras/ERK and PI3K/Akt pathways in non-small cell lung carcinoma (NSCLC) cell lines. EpoR synthesis is normally under the control of GATA-1, but NSCLC cells exhibit decreased GATA-1 levels compared to GATA-2, -3 and -6, suggesting that GATA-1 is not essential for EpoR production. The increased Epo-induced signalling was not associated with a growth advantage for the NSCLC cells.

Langerhans cells promote early germinal center formation in response to Leishmania-derived cutaneous antigens

Efficient formation of early GCs depends on the close interaction between GC B cells and antigen-primed CD4+ follicular helper T cells (TFH). A tight and stable formation of TFH/B cell conjugates is required for cytokine-driven immunoglobulin class switching and somatic hypermutation of GC B cells. Recently, it has been shown that the formation of TFH/B cell conjugates is crucial for B-cell differentiation and class switch following infection with Leishmania major parasites. However, the subtype of DCs responsible for TFH-cell priming against dermal antigens is thus far unknown. Utilizing a transgenic C57BL/6 mouse model designed to trigger the ablation of Langerin+ DC subsets in vivo, we show that the functionality of TFH/B cell conjugates is disturbed after depletion of Langerhans cells (LCs): LC-depleted mice show a reduction in somatic hypermutation in B cells isolated from TFH/B cell conjugates and markedly reduced GC reactions within skin-draining lymph nodes. In conclusion, this study reveals an indispensable role for LCs in promoting GC B-cell differentiation following cutaneous infection with Leishmania major parasites. We propose that LCs are key regulators of GC formation and therefore have broader implications for the development of allergies and autoimmunity as well as for future vaccination strategies.

Expression of transketolase-like 1 protein (TKTL1) in human endometrial cancer

Malignant tumors metabolize glucose to lactate even in the presence of oxygen (aerobic glycolysis). The metabolic switch from oxidative glycolysis to non-oxidative fermentation of glucose and proteins performed by the tumor cells seems to be associated with TKTL1 and pAkt overexpression. Therefore the aim of the present study was to investigate the expression of TKTL1 and pAkt in human specimens of endometrial cancer as compared to benign endometrium. Additionally, expression of the glucose transporter GLUT1 was also investigated as aerobic glycolysis is associated with an increased need for glucose.

DEAD-box helicase DP103 defines metastatic potential of human breast cancers

Despite advancement in breast cancer treatment, 30% of patients with early breast cancers experience relapse with distant metastasis. It is a challenge to identify patients at risk for relapse; therefore, the identification of markers and therapeutic targets for metastatic breast cancers is imperative. Here, we identified DP103 as a biomarker and metastasis-driving oncogene in human breast cancers and determined that DP103 elevates matrix metallopeptidase 9 (MMP9) levels, which are associated with metastasis and invasion through activation of NF-κB. In turn, NF-κB signaling positively activated DP103 expression. Furthermore, DP103 enhanced TGF-β-activated kinase-1 (TAK1) phosphorylation of NF-κB-activating IκB kinase 2 (IKK2), leading to increased NF-κB activity. Reduction of DP103 expression in invasive breast cancer cells reduced phosphorylation of IKK2, abrogated NF-κB-mediated MMP9 expression, and impeded metastasis in a murine xenograft model. In breast cancer patient tissues, elevated levels of DP103 correlated with enhanced MMP9, reduced overall survival, and reduced survival after relapse. Together, these data indicate that a positive DP103/NF-κB feedback loop promotes constitutive NF-κB activation in invasive breast cancers and activation of this pathway is linked to cancer progression and the acquisition of chemotherapy resistance. Furthermore, our results suggest that DP103 has potential as a therapeutic target for breast cancer treatment.

Manganese Superoxide Dismutase Is a Promising Target for Enhancing Chemosensitivity of Basal-Like Breast Carcinoma

AIMS: Although earlier reports highlighted a tumor suppressor role for manganese superoxide dismutase (MnSOD), recent evidence indicates increased expression in a variety of human cancers including aggressive breast carcinoma. In the present article, we hypothesized that MnSOD expression is significantly amplified in the aggressive breast carcinoma basal subtype, and targeting MnSOD could be an attractive strategy for enhancing chemosensitivity of this highly aggressive breast cancer subtype.

RESULTS: Using MDA-MB-231 and BT549 as a model of basal breast cancer cell lines, we show that knockdown of MnSOD decreased the colony-forming ability and sensitized the cells to drug-induced cell death, while drug resistance was associated with increased MnSOD expression. In an attempt to develop a clinically relevant approach to down-regulate MnSOD expression in patients with basal breast carcinoma, we employed activation of the peroxisome proliferator-activated receptor gamma (PPARγ) to repress MnSOD expression; PPARγ activation significantly reduced MnSOD expression, increased chemosensitivity, and inhibited tumor growth. Moreover, as a proof of concept for the clinical use of PPARγ agonists to decrease MnSOD expression, biopsies derived from breast cancer patients who had received synthetic PPARγ ligands as anti-diabetic therapy had significantly reduced MnSOD expression. Finally, we provide evidence to implicate peroxynitrite as the mechanism involved in the increased sensitivity to chemotherapy induced by MnSOD repression.

INNOVATION AND CONCLUSION: These data provide evidence to link increased MnSOD expression with the aggressive basal breast cancer, and underscore the judicious use of PPARγ ligands for specifically down-regulating MnSOD to increase the chemosensitivity of this subtype of breast carcinoma.

KATNAL1 regulation of sertoli cell microtubule dynamics is essential for spermiogenesis and male fertility

Spermatogenesis is a complex process reliant upon interactions between germ cells (GC) and supporting somatic cells. Testicular Sertoli cells (SC) support GCs during maturation through physical attachment, the provision of nutrients, and protection from immunological attack. This role is facilitated by an active cytoskeleton of parallel microtubule arrays that permit transport of nutrients to GCs, as well as translocation of spermatids through the seminiferous epithelium during maturation. It is well established that chemical perturbation of SC microtubule remodelling leads to premature GC exfoliation demonstrating that microtubule remodelling is an essential component of male fertility, yet the genes responsible for this process remain unknown. Using a random ENU mutagenesis approach, we have identified a novel mouse line displaying male-specific infertility, due to a point mutation in the highly conserved ATPase domain of the novel KATANIN p60-related microtubule severing protein Katanin p60 subunit A-like1 (KATNAL1). We demonstrate that Katnal1 is expressed in testicular Sertoli cells (SC) from 15.5 days post-coitum (dpc) and that, consistent with chemical disruption models, loss of function of KATNAL1 leads to male-specific infertility through disruption of SC microtubule dynamics and premature exfoliation of spermatids from the seminiferous epithelium. The identification of KATNAL1 as an essential regulator of male fertility provides a significant novel entry point into advancing our understanding of how SC microtubule dynamics promotes male fertility. Such information will have resonance both for future treatment of male fertility and the development of non-hormonal male contraceptives.

Gene expression and epigenetic discovery screen reveal methylation of SFRP2 in prostate cancer

Aberrant activation of Wnts is common in human cancers, including prostate. Hypermethylation associated transcriptional silencing of Wnt antagonist genes SFRPs (Secreted Frizzled-Related Proteins) is a frequent oncogenic event. The significance of this is not known in prostate cancer. The objectives of our study were to (i) profile Wnt signaling related gene expression and (ii) investigate methylation of Wnt antagonist genes in prostate cancer. Using TaqMan Low Density Arrays, we identified 15 Wnt signaling related genes with significantly altered expression in prostate cancer; the majority of which were upregulated in tumors. Notably, histologically benign tissue from men with prostate cancer appeared more similar to tumor (r = 0.76) than to benign prostatic hyperplasia (BPH; r = 0.57, p < 0.001). Overall, the expression profile was highly similar between tumors of high (≥ 7) and low (≤ 6) Gleason scores. Pharmacological demethylation of PC-3 cells with 5-Aza-CdR reactivated 39 genes (≥ 2-fold); 40% of which inhibit Wnt signaling. Methylation frequencies in prostate cancer were 10% (2/20) (SFRP1), 64.86% (48/74) (SFRP2), 0% (0/20) (SFRP4) and 60% (12/20) (SFRP5). SFRP2 methylation was detected at significantly lower frequencies in high-grade prostatic intraepithelial neoplasia (HGPIN; 30%, (6/20), p = 0.0096), tumor adjacent benign areas (8.82%, (7/69), p < 0.0001) and BPH (11.43% (4/35), p < 0.0001). The quantitative level of SFRP2 methylation (normalized index of methylation) was also significantly higher in tumors (116) than in the other samples (HGPIN = 7.45, HB = 0.47, and BPH = 0.12). We show that SFRP2 hypermethylation is a common event in prostate cancer. SFRP2 methylation in combination with other epigenetic markers may be a useful biomarker of prostate cancer.

Docetaxel maintains its cytotoxic activity under hypoxic conditions in prostate cancer cells

OBJECTIVE: The efficacy of docetaxel has recently been shown to be increased under hypoxic conditions through the down-regulation of hypoxia-inducible-factor 1α (HIF1A). Overexpression of the hypoxia-responsive gene class III β-tubulin (TUBB3) has been associated with docetaxel resistance in a number of cancer models. We propose that administration of docetaxel to prostate patients has the potential to reduce the hypoxic response through HIF1A down-regulation and that TUBB3 down-regulation participates in sensitivity to docetaxel.

METHODS: The cytotoxic effect of docetaxel was determined in both 22Rv1 and DU145 prostate cancer cell lines and correlated with HIF1A expression levels under aerobic and hypoxic conditions. Hypoxia-induced chemoresistance was investigated in a pair of isogenic docetaxel-resistant PC3 cell lines. Basal and hypoxia-induced TUBB3 gene expression levels were determined and correlated with methylation status at the HIF1A binding site.

RESULTS: Prostate cancer cells were sensitive to docetaxel under both aerobic and hypoxic conditions. Hypoxic cytotoxicity of docetaxel was consistent with a reduction in detected HIF1A levels. Sensitivity correlated with reduced basal and hypoxia-induced HIF1A and TUBB3 expression levels. The TUBB3 HIF1A binding site was hypermethylated in prostate cell lines and tumor specimens, which may exclude transcription factor binding and induction of TUBB3 expression. However, acquired docetaxel resistance was not associated with TUBB3 overexpression.

CONCLUSION: These data suggest that the hypoxic nature of a tumor may have relevance as regard to their response to docetaxel. Further investigation into the nature of this relationship may allow identification of novel targets to improve tumor control in prostate cancer patients.

Microtubule-targeting-compound PBOX-15 radiosensitizes cancer cells in vitro

BACKGROUND: We proposed to investigate the radiosensitizing properties of PBOX-15, a novel microtubule-disrupting agent, in a panel of cancer cell lines.

RESULTS: PBOX-15 treatment was associated with significant cell kill and increased radiosensitivity in all three cell lines tested. The number of surviving cells in response to the combined treatment was significantly less than PBOX -15 alone in 22Rv1 cells. In these cells, radiosensitisation correlated with induction of G2/M cell cycle arrest by PBOX-15. The compound sustained its activity and increased HIF-1Α expression under hypoxic conditions. PBOX-15 prevented onset of hypoxia-induced radioresistance in hypoxic prostate cells and reduced the surviving fraction of irradiated hypoxic cells to levels similar to those achieved under aerobic conditions.

METHODS: Clonogenic assays were used to determine sensitivity of a panel of cancer cell lines (22Rv1, A549, U87) to PBOX-15 alone or in combination with a single 2Gy dose fraction. Induction of cell cycle arrest and apoptosis was investigated in 22Rv1 prostate cancer cells. The cytotoxic properties of the compound under hypoxic conditions were correlated with Hypoxia Inducible Factor 1 alpha (HIF-1Α) gene and protein expression levels and its radiosensitisation potential was investigated in hypoxic 22Rv1 using clonogenic assays.

CONCLUSIONS: This preliminary data identifies the potential of PBOX-15 as a novel radiosensitising agent for the management of solid tumours and eradication of hypoxic cells.

In silico analysis and DHPLC screening strategy identifies novel apoptotic gene targets of aberrant promoter hypermethylation in prostate cancer

BACKGROUND: Aberrant DNA methylation has been implicated as a key survival mechanism in cancer, whereby promoter hypermethylation silences genes essential for many cellular processes including apoptosis. Limited data is available on the methylation profile of apoptotic genes in prostate cancer (CaP). The aim of this study was to profile methylation of apoptotic-related genes in CaP using denaturing high performance liquid chromatography (DHPLC).

METHODS: Based on an in silico selection process, 13 genes were screened for methylation in CaP cell lines using DHPLC. Quantitative methylation specific PCR was employed to determine methylation levels in prostate tissue specimens (n = 135), representing tumor, histologically benign prostate, high-grade prostatic intraepithelial neoplasia and benign prostatic hyperplasia. Gene expression was measured by QRT-PCR in cell lines and tissue specimens.

RESULTS: The promoters of BIK, BNIP3, cFLIP, TMS1, DCR1, DCR2, and CDKN2A appeared fully or partially methylated in a number of malignant cell lines. This is the first report of aberrant methylation of BIK, BNIP3, and cFLIP in CaP. Quantitative methylation analysis in prostate tissues identified 5 genes (BNIP3, CDKN2A, DCR1, DCR2 and TMS1) which were frequently methylated in tumors but were unmethylated in 100% of benign tissues. Furthermore, 69% of tumors were methylated in at least one of the five-gene panel. In the case of all genes, except BNIP3, promoter hypermethylation was associated with concurrent downregulation of gene expression.

CONCLUSION: Future examination of this "CaP apoptotic methylation signature" in a larger cohort of patients is justified to further evaluate its value as a diagnostic and prognostic marker.

Novel microtubule-targeting agents, pyrrolo-1,5-benzoxazepines, induce apoptosis in multi-drug-resistant cancer cells

PURPOSE: The development of multi-drug resistance (MDR) due to the expression of members of the ATP binding cassette (ABC) transporter family is a major obstacle in cancer treatment. The broad range of substrate specificities associated with these transporters leads to the efflux of many anti-cancer drugs from tumour cells. Therefore, the development of new chemotherapeutic agents that are not substrates of these transporters is important. We have recently demonstrated that some members of a novel series of pyrrolo-1,5-benzoxazepine (PBOX) compounds are microtubule-depolymerising agents that potently induce apoptosis in several cancer cell lines and impair growth of mouse breast tumours. The aim of this current study was to establish whether PBOXs were capable of inducing apoptosis in cancer cells expressing either P-glycoprotein or breast cancer resistance protein (BCRP), two of the main ABC transporters associated with MDR.

METHODS: We performed in vitro studies to assess the effects of PBOXs on cell proliferation, cell cycle and apoptosis in human cancer cell lines and their drug-resistant substrains expressing either P-glycoprotein or BCRP. In addition, we performed a preliminary molecular docking study to examine interactions between PBOXs and P-glycoprotein.

RESULTS: We established that three representative PBOXs, PBOX-6, -15 and -16 were capable of inducing apoptosis in drug-resistant HL60-MDR1 cells (expressing P-glycoprotein) and HL60-ABCG2 cells (expressing BCRP) with similar potencies as in parental human promyelocytic leukaemia HL60 cells. Likewise, resistance to PBOX-6 and -16 was not evident in P-glycoprotein-expressing A2780-ADR cells in comparison with parent human ovarian carcinoma A2780 cells. Finally, we deduced by molecular docking that PBOX-6 is not likely to form favourable interactions with the substrate binding site of P-glycoprotein.

CONCLUSION: Our results suggest that pro-apoptotic PBOX compounds may be potential candidates for the treatment of P-glycoprotein- or BCRP-associated MDR cancers.

Recognition of O6MeG lesions by MGMT and mismatch repair proficiency may be a prerequisite for low-dose radiation hypersensitivity

Low-dose hyper-radiosensitivity (HRS) is the phenomenon whereby cells exposed to radiation doses of less than approximately 0.5 Gy exhibit increased cell killing relative to that predicted from back-extrapolating high-dose survival data using a linear-quadratic model. While the exact mechanism remains to be elucidated, the involvement of several molecular repair pathways has been documented. These processes in turn are also associated with the response of cells to O6-methylguanine (O6MeG) lesions. We propose a model in which the level of low-dose cell killing is determined by the efficiency of both pre-replicative repair by the DNA repair enzyme O6-methylguanine methyltransferase (MGMT) and post-replicative repair by the DNA mismatch repair (MMR) system. We therefore hypothesized that the response of cells to low doses of radiation is dependent on the expression status of MGMT and MMR proteins. MMR (MSH2, MSH6, MLH1, PMS1, PMS2) and MGMT protein expression signatures were determined in a panel of normal (PWR1E, RWPE1) and malignant (22RV1, DU145, PC3) prostate cell lines and correlated with clonogenic survival and cell cycle analysis. PC3 and RWPE1 cells (HRS positive) were associated with MGMT and MMR proficiency, whereas HRS negative cell lines lacked expression of at least one (MGMT or MMR) protein. MGMT inactivation had no significant effect on cell survival. These results indicate a possible role for MMR-dependent processing of damage produced by low doses of radiation.