Abstract PCTAIRE1 is distant relative of the cyclin-dependent kinase family that has been implicated in spermatogenesis and neuronal development, but it has not been studied in cancer. Here, we report that PCTAIRE1 is expressed in prostate, breast, and cervical cancer cells, where its RNAi-mediated silencing causes growth inhibition with aberrant mitosis due to defects in centrosome dynamics. PCTAIRE1 was not similarly involved in proliferation of nontransformed cells, including diploid human IMR-90 fibroblasts. Through yeast two-hybrid screening, we identified tumor suppressor p27 as a PCTAIRE1 interactor. In vitro kinase assays showed PCTAIRE1 phosphorylates p27 at Ser10. PCTAIRE1 silencing modulated Ser10 phosphorylation on p27 and led to its accumulation in cancer cells but not in nontransformed cells. In a mouse xenograft model of PPC1 prostate cancer, conditional silencing of PCTAIRE1 restored p27 protein expression and suppressed tumor growth. Mechanistic studies in HeLa cells showed that PCTAIRE1 phosphorylates p27 during the S and M phases of the cell cycle. Notably, p27 silencing was sufficient to rescue cells from mitotic arrest caused by PCTAIRE1 silencing. Clinically, PCTAIRE1 was highly expressed in primary breast and prostate tumors compared with adjacent normal epithelial tissues. Together our findings reveal an unexpected role for PCTAIRE1 in regulating p27 stability, mitosis, and tumor growth, suggesting PCTAIRE1 as a candidate cancer therapeutic target. Cancer Res; 74(20); 5795–807. ©2014 AACR.

Supplementary Data from Expression of p21 Protein Predicts Clinical Outcome in DLBCL Patients Older than 60 Years Treated with R-CHOP but not CHOP: A Prospective ECOG and Southwest Oncology Group Correlative Study on E4494

Supplementary Figures 1-4 from Apoptotic Activity and Mechanism of 2-Cyano-3,12-Dioxoolean-1,9-Dien-28-Oic-Acid and Related Synthetic Triterpenoids in Prostate Cancer

Multiplex arrays designed for enzyme-linked immunosorbent assays (ELISAs) are robust and cost-effective for profiling biomarkers. Identification of relevant biomarkers in biological matrices or fluids helps in the understanding of disease pathogenesis. Here, we describe a sandwich ELISA-based multiplex assay to assess growth factor and cytokine levels in cerebrospinal fluid (CSF) samples derived from multiple sclerosis patients, amyotrophic lateral sclerosis patients, and control subjects without any neurological disorder. Results indicate that multiplex assay designed for the sandwich ELISA method is a unique, robust, and cost-effective method for profiling growth factors and cytokines present in CSF samples.

Supplementary Figures and Tables. Supplementary Fig. S1. Knockdown of PCTAIRE1 in PPC1 cells. Supplementary Fig. S2. Knockdown of PCTAIRE1 inhibits growth of Du145, MDA-MB-468 and HeLa cells. Supplementary Fig. S3. PCTAIRE1 regulates centrosome dynamics. Supplementary Fig. S4. PCTAIRE1 phosphorylates p27 at Ser10. Supplementary Fig. S5. PCTAIRE1 knockdown leads to accumulation of p27. Supplementary Fig. S6. Apoptosis induced by PCTAIRE1 knockdown, and subcellular localization of Eg5 in PCTAIRE1 knockdown cancer cells. Supplementary Fig. S7. Characterization of rabbit anti-PCTAIRE1 antibody and correlation of PCTAIRE1 and p27 expression. Supplementary Fig. S8. PCTAIRE1 expression is upregulated in cancers, and high PCTAIRE1 levels correlate with poor patient prognosis. Supplementary Table S1. Specific interaction of PCTAIRE1 with p27.

Supplementary Materials and Methods. Primers for quantitative RT-PCR. In vitro kinase assays. Time-lapse microscopy. Patient specimens. Gene expression. Statistical Analysis.

Supplementary Figures S1-S2 and Supplementary Table S1.

Growth factors and cytokines play a critical role in the pathogenesis of multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). The shift in the balance between pro-inflammatory and anti-inflammatory cytokines contributes toward autoimmunity. Epidermal growth factor (EGF) and fibroblast growth factor (FGF) are elevated in the cerebrospinal fluid (CSF) of MS and ALS patients. Identification of relevant biomarkers helps in the understanding of the disease pathogenesis. The goal is to establish a sandwich ELISA-based multiplex assay to assess growth factor and cytokine levels in CSF samples from MS and ALS patients. Capture antibodies against EGF, FGF, IFN-gamma, IL-6, IL-1beta and IL-4 imprinted in a multiplex array format into each well of a 96-well plate. Arrays subjected to multiplex sandwich ELISA of CSF samples. Test samples included CSF from Relapsing-Remitting MS (RRMS, N = 10), Secondary Progressive MS (SPMS, N = 10) and ALS patients [N=10]. Control samples included CSF from healthy subjects [N=10]. Colorimetric read-outs from multiplex ELISA are quantified using ImageJ analysis of standards and samples. Results suggest that multiplex array designed for ELISA is a unique, robust, and a cost-effective method for profiling growth factors and cytokines present in biological matrices. [*In memoriam: abstract submitted as a tribute to honor Dr. Barbara Tomik for her valuable contributions towards the collection and preparation of patient samples].

PCTAIRE1 is distant relative of the cyclin-dependent kinase family that has been implicated in spermatogenesis and neuronal development, but it has not been studied in cancer. Here, we report that PCTAIRE1 is expressed in prostate, breast, and cervical cancer cells, where its RNAi-mediated silencing causes growth inhibition with aberrant mitosis due to defects in centrosome dynamics. PCTAIRE1 was not similarly involved in proliferation of nontransformed cells, including diploid human IMR-90 fibroblasts. Through yeast two-hybrid screening, we identified tumor suppressor p27 as a PCTAIRE1 interactor. In vitro kinase assays showed PCTAIRE1 phosphorylates p27 at Ser10. PCTAIRE1 silencing modulated Ser10 phosphorylation on p27 and led to its accumulation in cancer cells but not in nontransformed cells. In a mouse xenograft model of PPC1 prostate cancer, conditional silencing of PCTAIRE1 restored p27 protein expression and suppressed tumor growth. Mechanistic studies in HeLa cells showed that PCTAIRE1 phosphorylates p27 during the S and M phases of the cell cycle. Notably, p27 silencing was sufficient to rescue cells from mitotic arrest caused by PCTAIRE1 silencing. Clinically, PCTAIRE1 was highly expressed in primary breast and prostate tumors compared with adjacent normal epithelial tissues. Together our findings reveal an unexpected role for PCTAIRE1 in regulating p27 stability, mitosis, and tumor growth, suggesting PCTAIRE1 as a candidate cancer therapeutic target. Cancer Res; 74(20); 1–13. 2014 AACR.

Light microscopy allows for the inexpensive and fast detection of neuronal /glial cell demise and estimation of infarct and traumatic lesion volumes; the direct correlates of cell death. Quantitative assessment of brain tissue damage following stroke, traumatic brain injury (TBI ) or neurodegenerative diseases, and recovery after therapeutic intervention has been facilitated by recent developments in computer-assisted image analysis technologies that enable more objective and accurate morphometric quantification of cell injury in whole brain sections. In this chapter, the proposed workflow describes what tasks need to be fulfilled to visualize and gauge cell death characterization by histological stains and immunohistochemical markers.

Abstract Prostate cancer (PCa) is among the leading causes of cancer-related death in men. Androgen receptor (AR) signaling plays a seminal role in prostate development and homeostasis, and dysregulation of this pathway is intimately linked to prostate cancer pathogenesis and progression. Here, we identify the cytosolic NLR-related protein NWD1 as a novel modulator of AR signaling. We determined that expression of NWD1 becomes elevated during prostate cancer progression, based on analysis of primary tumor specimens. Experiments with cultured cells showed that NWD1 expression is up-regulated by the sex-determining region Y (SRY) proteins. Gene silencing by short hairpin RNA (shRNA), in conjunction with transcriptional profiling, showed that NWD1 is required for expression of PDEF (prostate-derived Ets factor), which is known to bind and co-regulate AR. Of note, NWD1 modulates AR protein levels. Silencing NWD1 in PCa cell lines reduces the levels of androgen-stimulated accumulation of nuclear AR and suppresses activity of androgen-driven reporter genes. NWD1 knockdown potently suppressed growth of androgen-dependent LNCaP prostate cancer cells, thus showing its functional importance in an AR-dependent tumor cell model. Proteomics analysis suggested that NWD1 associates with various molecular chaperones commonly related to AR complexes. Altogether, our data suggest a role for tumor-associated over-expression of NWD1 in the dysregulation of AR signaling in PCa. Citation Format: Ricardo G. Correa, Maryla Krajewska, Carl F. Ware, Motti Gerlic, John C. Reed. The NLR-related protein NWD1 is associated with prostate cancer and modulates androgen receptor signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-74. doi:10.1158/1538-7445.AM2014-LB-74

Prostate cancer (PCa) is among the leading causes of cancer-related death in men. Androgen receptor (AR) signaling plays a seminal role in prostate development and homeostasis, and dysregulation of this pathway is intimately linked to prostate cancer pathogenesis and progression. Here, we identify the cytosolic NLR-related protein NWD1 as a novel modulator of AR signaling. We determined that expression of NWD1 becomes elevated during prostate cancer progression, based on analysis of primary tumor specimens. Experiments with cultured cells showed that NWD1 expression is up-regulated by the sex-determining region Y (SRY) family proteins. Gene silencing procedures, in conjunction with transcriptional profiling, showed that NWD1 is required for expression of PDEF (prostate-derived Ets factor), which is known to bind and co-regulate AR. Of note, NWD1 modulates AR protein levels. Depleting NWD1 in PCa cell lines reduces AR levels and suppresses activity of androgen-driven reporter genes. NWD1 knockdown potently suppressed growth of androgen-dependent LNCaP prostate cancer cells, thus showing its functional importance in an AR-dependent tumor cell model. Proteomic analysis suggested that NWD1 associates with various molecular chaperones commonly related to AR complexes. Altogether, these data suggest a role for tumor-associated over-expression of NWD1 in dysregulation of AR signaling in PCa.

PCTAIRE1 is distant relative of the cyclin-dependent kinase family that has been implicated in spermatogenesis and neuronal development, but it has not been studied in cancer. Here, we report that PCTAIRE1 is expressed in prostate, breast, and cervical cancer cells, where its RNAi-mediated silencing causes growth inhibition with aberrant mitosis due to defects in centrosome dynamics. PCTAIRE1 was not similarly involved in proliferation of nontransformed cells, including diploid human IMR-90 fibroblasts. Through yeast two-hybrid screening, we identified tumor suppressor p27 as a PCTAIRE1 interactor. In vitro kinase assays showed PCTAIRE1 phosphorylates p27 at Ser10. PCTAIRE1 silencing modulated Ser10 phosphorylation on p27 and led to its accumulation in cancer cells but not in nontransformed cells. In a mouse xenograft model of PPC1 prostate cancer, conditional silencing of PCTAIRE1 restored p27 protein expression and suppressed tumor growth. Mechanistic studies in HeLa cells showed that PCTAIRE1 phosphorylates p27 during the S and M phases of the cell cycle. Notably, p27 silencing was sufficient to rescue cells from mitotic arrest caused by PCTAIRE1 silencing. Clinically, PCTAIRE1 was highly expressed in primary breast and prostate tumors compared with adjacent normal epithelial tissues. Together our findings reveal an unexpected role for PCTAIRE1 in regulating p27 stability, mitosis, and tumor growth, suggesting PCTAIRE1 as a candidate cancer therapeutic target.

Abstract Leukemia stem cells (LSC) play a crucial role in initiation, maintenance, and progression of chronic myeloid leukemia (CML). Targeted therapy with a BCR-ABL targeted tyrosine kinase inhibitor (e.g. dasatinib) has improved survival of patients with chronic phase CML. However, like blast crisis (BC) CML, survival remains only 30% over 5 years, and treatment frequently fails to eliminate dormant LSC that are hypothesized to drive CML relapse. Overexpression of BCL2 family genes has been observed in human BC CML and may fuel LSC survival. Our previous data demonstrated that pro-survival long isoforms of BCL2 and MCL1 were preferentially expressed in BC CML LSC compared to normal stem cells. These data showed that the bone marrow (BM) is enriched for BCL2 and MCL1-expressing LSC and serves as a reservoir for therapeutic resistance, thereby underscoring the importance of pan-BCL2 inhibition in the BM. A potent pan-BCL2 inhibitor, sabutoclax, has been identified that inhibits the binding of BH3 peptides to Bcl-XL, Bcl-2, Mcl-1 and Bfl1-1 at nanomolar concentrations. To demonstrate whether sabutoclax or the combination of sabutoclax and dasatinib could synergize to eradicate LSC in the BM, humanized BC CML LSC mouse models were established by using 50,000 CD34+ cells selected from primary BC CML patient samples and transplanted into neonatal RAG2-/-γc-/- mice. Mice were screened for peripheral blood (PB) engraftment of human cells at 6 weeks, randomized, and dosed with (1) vehicle, (2) sabutoclax (10mg/kg, i.v., twice a week for 2 weeks), (3) dasatinib (50mg/kg, p.o., daily for 2 weeks), or (4) combination. Mice were sacrificed one day after last dose; PB, spleen (SP) and BM were collected for FACS analysis to measure engraftment and cell cycle status of LSC in different hematopoietic niches. BCL2 and MCL1 expression was measured in BM and SP by isoform specific qRT-PCR and immunohistochemistry. Treatment of CML LSC-transplanted mice with sabutoclax alone led to a significant reduction in LSC burden in the SP, but only slightly in the BM. Meanwhile, Single agent sabutoclax reduced the engraftment of quiescent LSC. The effect of sabutoclax synergized with dasatinib to further reduce LSC burden in the BM to eradicate LSC that drive therapeutic resistance. Importantly, these treatments spare normal human stem cells in both BM and SP thereby providing in vivo evidence of a reasonable therapeutic index. Sabutoclax not only reduced the expression of BCL2 and MCL1 mRNA long isoform in the BM, but also significantly reduced the number of human CD34+, BCL2+ and MCL1+ cells in both BM and SP. These results demonstrate that BM and SP niche BC CML LSC survival is driven by overexpression of multiple pro-survival BCL2 family isoforms rendering them susceptible to a novel pan-BCL2 inhibitor, sabutoclax, at doses that spare normal hematopoietic progenitors. Citation Format: Wenxue Ma, Janine Low-Marchelli, Heather S. Leu, Angela Court Recart, Jun Wei, Xianshu Huang, Maryla Krajewska, Maurizio Pellecchia, John C. Reed, Catriona H.M. Jamieson. BCR-ABL tyrosine kinase inhibition combined with a novel pan BCL2 family inhibitor abrogates leukemia stem cell survival in a niche-dependent manner. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 217. doi:10.1158/1538-7445.AM2013-217

The complexity of survival mechanisms in cancer cells from patients remains poorly understood. To obtain a comprehensive picture of tumour cell survival in lethal prostate cancer metastases, we examined five survival proteins that operate within three survival pathways in a cohort of 185 lethal metastatic prostate metastases obtained from 44 patients. The expression levels of BCL-2, BCL-XL, MCL-1, cytoplasmic survivin, nuclear survivin, and stathmin were measured by immunohistochemistry in a tissue microarray. Simultaneous expression of three or more proteins occurred in 81% of lethal prostate cancer metastases and BCL-2, cytoplasmic survivin and MCL-1 were co-expressed in 71% of metastatic sites. An unsupervised cluster analysis separated bone and soft tissue metastases according to patterns of survival protein expression. BCL-2, cytoplasmic survivin and MCL-1 had significantly higher expression in bone metastases (p < 10(-5)), while nuclear survivin was significantly higher in soft tissue metastases (p = 3 × 10(-14)). BCL-XL overexpression in soft tissue metastases almost reached significance (p = 0.09), while stathmin expression did not (p = 0.28). In addition, the expression of MCL-1 was significantly higher in AR-positive tumours. Neuroendocrine differentiation was not associated with specific survival pathways. These studies show that bone and soft tissue metastases from the same patient differ significantly in expression of a panel of survival proteins and that with regard to survival protein expression, expression is associated with the metastatic site and not the patient. Altogether, this suggests that optimal therapeutic inhibition may require combinations of drugs that target both bone and soft tissue-specific survival pathways.

Host innate immune responses to DNA viruses involve members of the nucleotide-binding domain, leucine-rich repeat and pyrin domain containing protein (NLRP) family, which form “inflammasomes” that activate caspase-1, resulting in proteolytic activation of cytokines interleukin (IL)-1β and IL-18. We hypothesized that DNA viruses would target inflammasomes to overcome host defense. A Vaccinia virus (VACV) B-cell CLL/lymphoma 2 (Bcl-2) homolog, F1L, was demonstrated to bind and inhibit the NLR family member NLRP1 in vitro. Moreover, infection of macrophages in culture with virus lacking F1L (ΔF1L) caused increased caspase-1 activation and IL-1β secretion compared with wild-type virus. Virulence of ΔF1L virus was attenuated in vivo, causing altered febrile responses, increased proteolytic processing of caspase-1, and more rapid inflammation in lungs of infected mice without affecting cell death or virus replication. Furthermore, we found that a hexapeptide from F1L is necessary and sufficient for inhibiting the NLRP1 inflammasome in vitro, thus identifying a peptidyl motif required for binding and inhibiting NLRP1. The functional importance of this NLRP1-binding motif was further confirmed by studies of recombinant ΔF1L viruses reconstituted either with the wild-type F1L or a F1L mutant that fails to bind NLRP1. Cellular infection with wild-type F1L reconstituted virus-suppressed IL-1β production, whereas mutant F1L did not. In contrast, both wild-type and mutant versions of F1L equally suppressed apoptosis. In vivo, the NLR nonbinding F1L mutant virus exhibited an attenuated phenotype similar to ΔF1L virus, thus confirming the importance of F1L interactions with NLRP1 for viral pathogenicity in mice. Altogether, these findings reveal a unique viral mechanism for evading host innate immune responses.

Cancer of the ovary confers the worst prognosis among women with gynecological malignancies, primarily because most ovarian cancers are diagnosed at late stage. Hence, there is a substantial need to develop new diagnostic biomarkers to enable detection of ovarian cancer at earlier stages, which would confer better prognosis. In addition, the identification of druggable targets is of substantial interest to find new therapeutic strategies for ovarian cancer.

Leukemia stem cells (LSCs) play a pivotal role in the resistance of chronic myeloid leukemia (CML) to tyrosine kinase inhibitors (TKIs) and its progression to blast crisis (BC), in part, through the alternative splicing of self-renewal and survival genes. To elucidate splice-isoform regulators of human BC LSC maintenance, we performed whole-transcriptome RNA sequencing, splice-isoform-specific quantitative RT-PCR (qRT-PCR), nanoproteomics, stromal coculture, and BC LSC xenotransplantation analyses. Cumulatively, these studies show that the alternative splicing of multiple prosurvival BCL2 family genes promotes malignant transformation of myeloid progenitors into BC LSCS that are quiescent in the marrow niche and that contribute to therapeutic resistance. Notably, sabutoclax, a pan-BCL2 inhibitor, renders marrow-niche-resident BC LSCs sensitive to TKIs at doses that spare normal progenitors. These findings underscore the importance of alternative BCL2 family splice-isoform expression in BC LSC maintenance and suggest that the combinatorial inhibition of prosurvival BCL2 family proteins and BCR-ABL may eliminate dormant LSCs and obviate resistance.

Autophagy is a lysosomal degradation pathway that converts macromolecules into substrates for energy production during nutrient-scarce conditions such as those encountered in tumor microenvironments. Constitutive mitochondrial uptake of endoplasmic reticulum (ER) Ca 2+ mediated by inositol triphosphate receptors (IP 3 Rs) maintains cellular bioenergetics, thus suppressing autophagy. We show that the ER membrane protein Bax inhibitor-1 (BI-1) promotes autophagy in an IP 3 R-dependent manner. By reducing steady-state levels of ER Ca 2+ via IP 3 Rs, BI-1 influences mitochondrial bioenergetics, reducing oxygen consumption, impacting cellular ATP levels, and stimulating autophagy. Furthermore, BI-1-deficient mice show reduced basal autophagy, and experimentally reducing BI-1 expression impairs tumor xenograft growth in vivo. BI-1's ability to promote autophagy could be dissociated from its known function as a modulator of IRE1 signaling in the context of ER stress. The results reveal BI-1 as a novel autophagy regulator that bridges Ca 2+ signaling between ER and mitochondria, reducing cellular oxygen consumption and contributing to cellular resilience in the face of metabolic stress.