Marked Reduction In Proliferation Research Articles

Turning Nonselective Inhibitors of Type I Protein Arginine Methyltransferases into Potent and Selective Inhibitors of Protein Arginine Methyltransferase 4 through a Deconstruction-Reconstruction and Fragment-Growing Approach.

Protein arginine methyltransferases (PRMTs) are important therapeutic targets, playing a crucial role in the regulation of many cellular processes and being linked to many diseases. Yet, there is still much to be understood regarding their functions and the biological pathways in which they are involved, as well as on the structural requirements that could drive the development of selective modulators of PRMT activity. Here we report a deconstruction–reconstruction approach that, starting from a series of type I PRMT inhibitors previously identified by us, allowed for the identification of potent and selective inhibitors of PRMT4, which regardless of the low cell permeability show an evident reduction of arginine methylation levels in MCF7 cells and a marked reduction of proliferation. We also report crystal structures with various PRMTs supporting the observed specificity and selectivity.

Dopamine regulates adult neurogenesis in the ventricular-subventricular zone via dopamine D3 angiotensin type 2 receptor interactions.

Adult neurogenesis is a dynamic and highly regulated process, and different studies suggest that dopamine modulates ventricular-subventricular zone (V-SVZ) neurogenesis. However, the specific role of dopamine and the mechanisms/factors underlying its effects on physiological and pathological conditions such as Parkinson's disease (PD) are not fully understood. Recent studies have described counter-regulatory interactions between renin-angiotensin system (RAS) and dopamine in peripheral tissues and in the nigrostriatal system. We have previously demonstrated that angiotensin receptors regulate proliferation and generation of neuroblasts in the rodent V-SVZ. However, possible interactions between dopamine receptors and RAS in the V-SVZ and their role in alterations of neurogenesis in animal models of PD have not been investigated. In V-SVZ cultures, activation of dopamine receptors induced changes in the expression of angiotensin receptors. Moreover, dopamine, via D2-like receptors and particularly D3 receptors, increased generation of neurospheres derived from the V-SVZ and this effect was mediated by angiotensin type-2 (AT2) receptors. In rats, we observed a marked reduction in proliferation and generation of neuroblasts in the V-SVZ of dopamine-depleted animals, and inhibition of AT1 receptors or activation of AT2 receptors restored proliferation and generation of neuroblasts to control levels. Moreover, intrastriatal mesencephalic grafts partially restored proliferation and generation of neuroblasts observed in the V-SVZ of dopamine-depleted rats. Our data revealed that dopamine and angiotensin receptor interactions play a major role in the regulation of V-SVZ and suggest potential beneficial effects of RAS modulators on the regulation of adult V-SVZ neurogenesis.

Abstract P4-06-02: Multi-parameter FACS sorting identifies higher mutational burden in aromatase inhibitor resistant subclones in estrogen positive breast cancer at diagnosis

Abstract Background: In many cancers, intratumor heterogeneity is associated with poor outcomes and resistance to treatment but datasets and techniques to identify genomic differences between the resistant and sensitive subclones in primary tumors have been limited. Short-term presurgical treatment of estrogen positive (ER+) breast cancer with aromatase inhibitors (AIs) leads to marked reduction in proliferation in most tumours. However, some proliferating cells remain in most ER+ cancers after treatment and these cells may be responsible for the outgrowth of resistant clones. We provide data from a novel approach that may allow a better understanding of the molecular drivers of the de novo endocrine resistant cells that may underpin the eventual emergence of clinical resistance and allow early intervention to avoid this. Methods: Twenty-six FFPE ER+ breast cancer excision biopsies that showed partial Ki67 suppression after 2-weeks neoadjuvant AI treatment were studied. Single cell suspensions were obtained by enzymatic dispersion of 50-µm thick sections using a collagenase/dispase digestion method and were simultaneously stained for cytokeratin (Alexa Fluor 488), vimentin (Alexa Fluor 647), and DNA (DAPI). The stained suspensions were analysed using BD FACSAria II flow cytometer, and cytokeratin positive (epithelial) cells were separated from vimentin positive (stromal) cells by gating on a dot plot showing green (Alexa Fluor 488) versus far red (Alexa Fluor 647) fluorescence. Epithelial cells were further subdivided according to cell cycle into G0/G1 (mainly non-proliferating) and S/G2M (proliferating) populations using DNA histogram gating based on DAPI staining. Cells were sorted until ~90,000 cells or all cells if <90,000 were collected for each sub-population and DNA was extracted for whole exome sequencing. Consensus calls from multiple software packages were used to detect somatic mutations in epithelial cells using the vimentin positive cells as normal controls. Results: Twenty-six samples were FACS sorted into the 3 populations (non-proliferating and proliferating epithelial cells and stromal cells). Vimentin/stromal populations were almost all diploid and most epithelial populations were aneuploid. Two samples had more than 2 epithelial peaks suggesting multiple tumor subclones. The mean coverage for all samples was 52x and mean number of mutations per sorted sample was 418 (missense, nonsense, splicing, frame shift and non-stop mutations). Two samples had low coverage and were removed for further analysis, and in the remaining 24 FACS sorted samples, mean somatic mutational burden was significantly greater in proliferating cells compared to non-proliferation cells (mean 474 vs. 372, p=0.008 Wilcoxon signed rank test) with 5 tumors showing gross increases. There was no significant difference between the percent of the genome with copy number gains or losses between proliferating and non-proliferating cells. There were no individual genes that had significantly higher mutation rates in proliferating cells after correction for multiple testing. Conclusions: FACS sorting of neoadjuvant AI treated samples allows the separation of de novo AI-resistant from sensitive cell populations and revealed higher mutational burden in the AI resistant populations at the time of disease presentation. Interrogation of the mutational differences between the proliferative and non-proliferative cells may allow identification of putative drivers of resistance in individual tumours. Additional samples are under analysis to determine whether common drivers can be identified. Citation Format: Eugene F Schuster, Lila Zabalgo, Charles M Perou, Mitch Dowsett. Multi-parameter FACS sorting identifies higher mutational burden in aromatase inhibitor resistant subclones in estrogen positive breast cancer at diagnosis [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-06-02.

Abstract 123: Age-Related Diminishment of the Subventricular Zone Cytogenic Response and Its Contributions to Motor Recovery After Cortical Infarcts

Stroke increases proliferation within the subventricular zone (SVZ) cytogenic niche and causes subsequent migration of newborn cells towards the site of injury. We investigated the functional consequences of age-related blunting of the SVZ cytogenic response to ischemia. We found that there was a marked reduction in proliferation and neural stem cell markers within the SVZ of middle aged (aged 12-16 months) versus young adult (aged 3-5 months) mice in the intact brain and after photothrombotic infarcts in motor cortex. Using an inducible, heritable lineage tracing system (Nestin-CreER T2 :: Ai14 mice) to quantify SVZ-derived neural precursor cells (NPCs) that migrated towards the infarct, we found that there was a considerable age-related reduction in the number of NPCs in peri-infarct cortex. These findings indicate a marked diminishment of SVZ NPC proliferation and migration after focal ischemia by middle age. Next, we assessed the contributions of the SVZ cytogenic response to recovery of skilled motor function. We used glial fibrillary acidic protein-thymidine kinase mice to conditionally ablate NPCs with ganciclovir administration. In young adult mice, NPC ablation significantly impaired recovery of motor performance on the single seed reaching task after motor cortical infarcts. By contrast, NPC ablation did not affect motor recovery in middle aged mice. Importantly, the magnitude of recovery was less in middle aged mice—regardless of NPC ablation—than in control young adult mice. Middle aged mice recovered similarly to young adult mice lacking NPCs. These results indicate that SVZ cytogenesis contributes to functional improvements after cortical infarcts and that the diminishment of the cytogenic response with age may be implicated in age-related worsening of outcome after stroke. Restoration of SVZ cytogenesis in aged animals might improve behavioral recovery.

Tumor Heterogeneity Underlies Differential Cisplatin Sensitivity in Mouse Models of Small-Cell Lung Cancer

SummarySmall-cell lung cancer is the most aggressive type of lung cancer, characterized by a remarkable response to chemotherapy followed by development of resistance. Here, we describe SCLC subtypes in Mycl- and Nfib-driven GEMM that include CDH1-high peripheral primary tumor lesions and CDH1-negative, aggressive intrapulmonary metastases. Cisplatin treatment preferentially eliminates the latter, thus revealing a striking differential response. Using a combined transcriptomic and proteomic approach, we find a marked reduction in proliferation and metabolic rewiring following cisplatin treatment and present evidence for a distinctive metabolic and structural profile defining intrinsically resistant populations. This offers perspectives for effective combination therapies that might also hold promise for treating human SCLC, given the very similar response of both mouse and human SCLC to cisplatin.

Piwil2 is reactivated by HPV oncoproteins and initiates cell reprogramming via epigenetic regulation during cervical cancer tumorigenesis.

The human papillomavirus (HPV) oncoproteins E6 and E7 are risk factors that are primarily responsible for the initiation and progression of cervical cancer, and they play a key role in immortalization and transformation by reprogramming differentiating host epithelial cells. It is unclear how cervical epithelial cells transform into tumor-initiating cells (TICs). Here, we observed that the germ stem cell protein Piwil2 is expressed in pre-cancerous and malignant lesions of the cervix and cervical cancer cell lines with the exception of the non-HPV-infected C33a cell line. Knockdown of Piwil2 by shRNA led to a marked reduction in proliferation and colony formation, in vivo tumorigenicity, chemo-resistance, and the proportion of cancer stem-like cells. In contrast, Piwil2 overexpression induced malignant transformation of HaCaT cells and the acquisition of tumor-initiating capabilities. Gene-set enrichment analysis revealed embryonic stem cell (ESC) identity, malignant biological behavior, and specifically, activation targets of the cell reprogramming factors c-Myc, Klf4, Nanog, Oct4, and Sox2 in Piwil2-overexpressing HaCaT cells. We further confirmed that E6 and E7 reactivated Piwil2 and that E6 and E7 overexpression resulted in a similar gene-set enrichment pattern as Piwil2 overexpression in HaCaT cells. Moreover, Piwil2 overexpression or E6 and E7 activation induced H3K9 acetylation but reduced H3K9 trimethylation, which contributed to the epigenetic reprogramming and ESC signature maintenance, as predicted previously. Our study demonstrates that Piwil2, reactivated by the HPV oncoproteins E6 and E7, plays an essential role in the transformation of cervical epithelial cells to TICs via epigenetics-based cell reprogramming.

Mechanisms of Action and Toxicity of the Mycotoxin Alternariol: A Review.

The mycotoxin alternariol (AOH) is produced by Alternaria fungi. It occurs naturally in foodstuffs and is frequently found as contaminant in fruit and grain products. Most information regarding AOH toxicity and the potential mechanisms involved comes from in vitro studies, as only very limited in vivo studies have been performed. AOH forms reactive oxygen species (ROS) and interacts with DNA topoisomerase, thereby generating both single (SSB)- and double-strand DNA beaks (DSB). This triggers various DNA damage response pathways. AOH causes a marked reduction in proliferation in mammalian cells due to cell cycle arrest often in the G2 /M-phase. After an additional inhibition of cytokinesis, cells with abnormal nuclei as well as polyploidy are reported. In macrophages, AOH may increase autophagic activity and induce senescence. Furthermore, AOH is found to change the morphology and phenotype of various human macrophage cell models. Studies so far indicate that the AOH-induced effects are primarily a result of DSB via its effects on topoisomerase activity. Thus, most probably there will be a threshold for the AOH-induced effects, typically seen in the 5-10 μM range. These in vitro mechanistic studies further support the in vivo studies suggesting low acute toxicity. However, a decreased immune response to infections and/or a disturbed balance of the adaptive immune system when exposed together with other mycotoxins cannot be excluded. This hypothesis needs to be further explored with proper in vivo studies.

Abstract 4263: Establishment and initial characterization of human hepatocellular carcinoma organoid culture

Abstract Background: Hepatocellular carcinoma (HCC) is the 3rd most leading cause of cancer death in the world. Despite extensive research, HCC has a strikingly bad prognosis due to the limited therapeutic options: Other than surgical resection, there is no curative treatment. No cytotoxic agent has shown efficacy so far, and the only currently approved systemic agent, sorafenib, is only used in a palliative context. This dilemma is due to the therapy-resistant biology as well as its variable pathogenesis and biological diversity, which requires excellent preclinical models to allow differential and innovative studies. Innovative 3D culture methods such as organoid culture have been increasingly implemented in routine in vitro research for many tumor entities. However, due to its distinct biology, organoid culture of HCC has been a challenge. We here describe the initial establishment and characterization of HCC organoid culture. Materials/Methods: Surgical HCC tissue specimens were obtained in the operating rooms of the University Hospital Dresden; biopsy specimens were obtained via CT-/ultrasound-guided biopsy of HCC lesions. All samples were processed according to a novel protocol and cultured in Matrigel supplemented with a highly specialized culture medium. Organoids were propagated, passaged and frozen at regular intervals. Proliferation was measured using a modified WST proliferation assay protocol. Next-generation sequencing (NGS) was performed on corresponding triplets of healthy tissue, tumor tissue and organoid samples using the TruSight One Sequencing Panel (Illumina) on an Illumina MiSeq Sequencer. Histology was performed on formalin-fixed and paraffin-embedded organoids using standard protocols. Results: We were able to establish a method to culture HCC samples as organoids in vitro. Proliferation assays revealed distinct differences in organoid growth of HCCs from different patients and correlated well with both clinical behavior and histological grading of the tumors. Amplicon sequencing (targeting 4,813 cancer-associated genes) of corresponding sets of tumor specimens and HCC organoids demonstrated the HCC origin of the organoids and their genetic and genomic stability in culture. Treatment of HCC organoids with sorafenib showed the expected reduction in proliferation and viability. Interestingly, treatment of the HCC organoids with hepatocyte growth factor (HGF) resulted in a marked reduction in proliferation as opposed to healthy liver organoids, which had increased proliferation activity upon stimulation with HGF. Histology of the HCC organoids showed distinct features of HCC and corresponded well with the corresponding HCC tissue samples. Conclusion: We successfully established a protocol reliably allowing 3D organoid culture of HCC. The organoids faithfully recapitulate the biology of the corresponding tumor and can be used for multiple purposes including in vitro therapeutic testing. Citation Format: Sebastian Schölch, Lahiri K. Nanduri, Daniel Kühn, Jürgen Weitz, Moritz Koch, Nuh N. Rahbari. Establishment and initial characterization of human hepatocellular carcinoma organoid culture. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4263.

Over-expression of telomere binding factors (TRF1 & TRF2) in renal cell carcinoma and their inhibition by using SiRNA induce apoptosis, reduce cell proliferation and migration invitro.

Telomere binding factors viz. TRF1 and TRF2 are a part of sheltrin complex that are present exclusively at the ends of chromosomes. These factors play an important role in maintaining chromosomal integrity at the ends. However, their status and role are not clear in renal cell carcinoma (RCC). Therefore, the present study was conducted to evaluate TRF1 and TRF2 expressions in RCC tissues. Further, the role of these factors involved in tumorigenesis was elucidated by gene silencing using siRNA in RCC cell line (A498). The present study documented a significant over-expression of TRF1 (P = 0.005) and TRF2 (P = 0.0048) mRNAs by real time PCR in RCC tissues as compared with adjacent normal kidney tissues. Immunohistochemistry studies also revealed higher expression of TRF1 and TRF2 proteins in RCC. Moreover, TRF1 or TRF2 gene silencing using siRNA showed marked reduction in proliferation of RCC cells (P = 0.000). Further, significantly induced cell cycle arrest (P = 0.000) and apoptosis of RCC cells (P = 0.000) was documented upon TRF1 or TRF2 gene silencing. Henceforth, the results deduce that TRF1 or TRF2 inhibitions play an important role in the induction of apoptosis in A498 cells, which may serve as a potential therapeutic target in RCC.

Synergistic Induction of Cell Death By Combined Inhibition of PIM and AKT Kinases in Cytogenetically Defined Standard and High-Risk Multiple Myeloma

The PIM and PI3K/Akt pathways have been studied extensively for their effects on cell growth, proliferation and survival and shown to have both distinct and overlapping functions. The PIM proteins (PIM1, 2 and 3) are serine/threonine kinases that are constitutively active, with control of PIM signaling largely at the level of transcriptional regulation. Recent studies have shown PIM2 mRNA to be highly expressed in multiple myeloma (MM) and Akt has previously been shown to be constitutively active in primary MM cells in at least 50% of samples. As results of targeting single signaling pathways can be compromised by compensatory mechanisms, we have investigated the effects of the pan-PIM inhibitor AZD1897 and the Akt inhibitor AZD5363 alone and in combination in MM, utilising a panel of cell lines representing the major MM translocation subtypes and primary patient CD138+ samples.Western blotting of a panel of human myeloma cell lines (HMCL) and primary MM samples showed that PIM2 is the major PIM isoform expressed. PIM2 protein levels were highest in HMCL with the (4;14) translocation. This finding was confirmed in primary samples where levels were quantified by densitometry (corrected for GAPDH expression) – relative levels in cases with t(4;14) 3.6±1, t(11;14) 1±0.4 and hyperdiploidy 0.7±0.3 (n=5, 6 and 4 respectively; p=0.02 by ANOVA).PIM and Akt inhibitors reduced proliferation in HMCL but had modest effects on cell death when used on their own. Combined PIM+Akt inhibition led to a more marked reduction in proliferation and produced synergistic cytotoxicity with induction of >50% cell death at 72 hours in 4 of 4 cell lines with t(4;14), 2 of 3 with t(14;16) and 2 of 4 with t(11;14). Chou-Talalay analysis confirmed synergistic effects with combination indices ranging from 0.1 to 0.8. Direct comparison of inhibitors of Akt, PI3K (GDC0941) and mTORC1/2 (KU-0063794) in combination with AZD1897 showed that the PIM+Akt inhibitor combination was most effective at inducing cell death in 8 out of 12 HMCL. As Akt is downstream of PI3K, this result may appear surprising – however, AZD5363 inhibits both Akt and has additional direct activity against p70S6K/S6K1. As previous studies have shown a PI3K-independent component to S6K signaling in some MM cells, this may at least partly explain this phenomenon.Investigation of downstream signaling showed that PIM inhibition alone led to decreased BAD S112 phosphorylation and of Akt alone to reduced FOXO3 and PRAS40 phosphorylation, confirming blockade of these validated targets at the concentrations employed. Combined PIM+Akt inhibition had enhanced effects compared with either agent alone on mTORC1 outputs, including phosphorylation of 4EBP1 and S6. Importantly, combined inhibition synergised to significantly reduce MCL1 levels in cells that showed marked cell death in response to AZD1897+AZD5363, but not in those that did not, suggesting a mechanistic basis for the cytotoxicity observed.Next we investigated the effect of PIM and Akt inhibition in 23 primary MM samples, subdivided by FISH analysis into standard risk [t(11;14), hyperdiploidy, normal] or adverse risk [t(4;14), t(14;16), gain of 1q, TP53 deleted] groups. Viable CD138+ cell survival after 72 hours in the standard risk group (n=13) was 74±4% of control for AZD1897(PIM inhibitor), 65±4% for AZD5363(Akt inhibitor) and 50±4% for the combination. The equivalent figures for the adverse risk group (n=10) were 70±4%, 57±8% and 38±6%, indicating that dual PIM and Akt targeting is effective across all clinical risk categories.We also assessed the effect of the combination of individual PIM or Akt inhibitors with other anti-MM agents. In HMCL treated with dexamethasone, enhanced effects were seen particularly for the AZD5363+Dex combination eg H929 cells, AZD5363 54% survival, Dex (10nM) 95%, both 15%; KMS28BM 59%, 90%, 28% respectively. Dual PIM+Akt inhibition could also enhance the effects of bortezomib in some HMCL eg H929 PIM+Akt 81% survival, BTZ (25nM) 73%, combination 28%.In conclusion, combined inhibition of PIM (AZD1897) and Akt (AZD5363) kinases resulted in significant anti-MM activity across all cytogenetically defined clinical risk groups, with synergistic induction of cell death. Biomarkers of activity included mTORC1 targets such as p4EBP1 and pS6 and also MCL1 levels. These results provide a rationale for clinical studies of combined PIM and Akt inhibition in MM. DisclosuresMeja:AstraZeneca: Research Funding. Huszar:AstraZeneca: Employment, Equity Ownership. Davies:AstraZeneca: Employment, Equity Ownership. Khwaja:AstraZeneca: Research Funding.

The plasminogen activator system: involvement in central nervous system inflammation and a potential site for therapeutic intervention

BackgroundExtracellular proteases such as plasminogen activators (PAs) and matrix metalloproteinases modulate cell-cell and cell-matrix interactions. Components of the PA/plasmin system have been shown to be increased in areas of inflammation, and have been suggested to play a role in inflammatory neurologic disorders such as epilepsy, stroke, brain trauma, Alzheimer's' disease and multiple sclerosis (MS). In the present study, we evaluated the involvement of the PA system in the animal model of MS, experimental autoimmune encephalomyelitis (EAE).MethodsEAE was induced by myelin oligodendrocyte glycoprotein (MOG) in mice deficient for the urokinase PA (uPA−/−), or the urokinase PA receptor (uPAR−/−). Mice were evaluated for EAE clinical signs and histopathologic parameters, and compared with wild-type (WT) EAE mice. Lymphocytes from the knockout (KO) and WT mice were analyzed for ex vivo restimulation, cytokine secretion, and antigen presentation. Finally, WT EAE mice were treated with PAI-1dp, an 18 amino acid peptide derived from the PA inhibitor protein (PAI-1).ResultsEAE was aggravated in uPA−/− and uPAR−/− mice, and this was accompanied by more severe histopathologic features and microglial activation. By contrast, specific T- cell reactivity towards the encephalitogenic antigen MOG was markedly reduced in the KO animals, as shown by a marked reduction in proliferation and pro-inflammatory cytokine secretion in these mice. Antigen presentation was also reduced in all the KO animals, raising an immunologic paradox. When the mice were treated with PAI-1, a peptide derived from the PA system, a marked and significant improvement in EAE was seen. The clinical improvement was linked to reduced T-cell reactivity, further emphasizing the importance of the PA system in immunomodulation during neuroinflammation.ConclusionsCumulatively, our results suggest a role for uPA and uPAR in EAE pathogenesis, as exacerbation of disease was seen in their absence. Furthermore, the successful amelioration of EAE by PAI-1 treatment suggests that the PA system can be considered a potential site for therapeutic intervention in the treatment of neuroimmune diseases.

Defective thymic progenitor development and mature T‐cell responses in a mouse model for Down syndrome

In addition to archetypal cognitive defects, Down syndrome (DS) is characterized by altered lymphocyte development and function, including premature thymic involution and increased incidence of infections. However, the potential mechanisms for these changes have not been fully elucidated. The current study used the Ts65Dn mouse model of DS to assess deficiencies in T-cell development and possible molecular alterations. Ts65Dn mice exhibited premature thymic involution and a threefold to fourfold decrease in the number and proportion of immature, double-negative thymocyte progenitors. In addition, there were twofold fewer double-positive and CD4 single-positive thymocytes in Ts65Dn thymuses. Reflecting this deficient thymic function, there were fewer naive T cells in the spleen and polyclonal stimulation of peripheral T cells exhibited a marked reduction in proliferation, suggesting a senescent phenotype. In contrast, B-cell progenitors were unchanged in the bone marrow of Ts65Dn mice, but in the spleen, there were decreased transitional and follicular B cells and these cells proliferated less upon antigen receptor stimulus but not in response to lipopolysaccharide. As a potential mechanism for diminished thymic function, immature thymocyte populations expressed diminished levels of the cytokine receptor interleukin-7Rα, which was associated with decreased proliferation and increased apoptosis. Increased oxidative stress and inhibition of the Notch pathway were identified as possible mediators of decreased interleukin-7Rα expression in Ts65Dn mice. The data suggest that immature thymocyte defects underlie immune dysfunction in DS and that increased oxidative stress and reduced cytokine signalling may alter lymphocyte development in Ts65Dn mice.

Abstract C6: Targeting Hsp90-dependent functional maturation of the androgen receptor in human prostate cancer

Abstract The molecular chaperone heat shock protein 90 (Hsp90) is an important target for cancer therapy as it is required for the correct maturation and function of its various client proteins, many of which are known oncogenes. In prostate cancer, targeting Hsp90 is particularly attractive as the androgen receptor (AR), the key mediator of prostate cancer cell growth and survival, depends on Hsp90 for its ligand-binding capacity and stability. Despite promising results in pre-clinical studies, to date Hsp90 inhibitors including 17-allylamino-demethoxygeldanamycin (17-AAG) have demonstrated limited efficacy in clinical trials for advanced prostate cancer. While this is in part due to drug formulation issues, we propose that a treatment strategy that simultaneously targets multiple aspects of AR action will more effectively eliminate AR-dependent prostate cancer cells than single agent treatment strategies. In this study, we markedly enhanced the efficacy of 17-AAG by combining lower doses of this agent with either an antiandrogen (bicalutamide) or a histone deacetylase inhibitor (vorinostat), both of which are inhibitors of AR function and/or expression. Interestingly, comparison of gene expression profiles altered by each combination indicated that these treatments did not markedly enhance abrogation of androgen signaling compared with individual agents. Rather, the combination treatments regulated expression of unique gene sets that were enriched for cell cycle, apoptosis, MAPK and insulin signaling. To further enhance the efficacy of a combinatorial approach involving Hsp90 inhibitors, we investigated two new generation Hsp90 inhibitors, NVP-AUY922 and the orally available NVP-HSP990. We found that both agents were significantly more potent with regards to modulation of Hsp90 client proteins, inhibition of cell proliferation and induction of cell death than 17-AAG in prostate cancer cell lines. To assess the combinatorial approach in human disease, we have developed a unique approach to study human prostate cancer where tumors are cultured as explants for up to 7 days with maintenance of tissue integrity, cell proliferation and androgen signaling. Using this ex vivo strategy, we observed that human prostate tumor tissue responds to the novel synthetic Hsp90 inhibitors, but not 17-AAG, with a marked reduction in proliferation. Importantly, these studies revealed dissociation between client protein modulation and proliferative response to Hsp90 inhibition, which was not seen in prostate cancer cell lines in vitro. Collectively, these findings suggest that these novel Hsp90 inhibitors warrant further clinical investigation in prostate cancer. Moreover, ex vivo tumor culture may better predict efficacy of these agents on an individual patient basis, and improve identification of markers of clinical response. Citation Format: Margaret M. Centenera, Sarah L. Carter, Joanna L. Gillis, Luke A. Selth, Peter D. Sutherland, Wayne D. Tilley, Lisa M. Butler. Targeting Hsp90-dependent functional maturation of the androgen receptor in human prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C6.

Abstract CN08-05: A combinatorial approach to targeting androgen receptor signaling in prostate cancer.

Abstract When androgen deprivation therapy (ADT) for prostate cancer fails and the disease progresses to a castrate-resistant state (castrate-resistant prostate cancer; CRPC), therapeutic options for patients are limited. It is now recognized that the majority of CRPC still express the androgen receptor (AR) and remain dependent on AR signaling for growth and survival. To limit growth or evolution of AR-dependent disease and thereby improve patient survival, we propose that a treatment strategy that simultaneously targets multiple features of the AR signaling axis will more effectively eliminate AR-dependent prostate cancer cells than single agent treatment strategies. We have shown that pharmacologically low doses of an AR antagonist (bicalutamide) in combination with either a heat shock protein 90 inhibitor (Hsp90i; 17-AAG) or a histone deacetylase inhibitor (vorinostat), results in synergistic growth suppression and induction of caspase-dependent death in AR-dependent prostate cancer cells. These drug combinations did not alter steady state protein levels of the AR, but suppressed AR activity, as evidenced by reduced prostate specific antigen levels. As expected, the drug combinations were not effective in androgen-independent, AR negative prostate cancer cells. To elucidate the mechanisms of drug synergy, we performed microarray studies to identify genetic programs modulated by each combination therapy or bicalutamide alone. Resultant data suggest that the combination treatments did not markedly enhance abrogation of androgen signaling compared with bicalutamide alone. Rather, the combination treatments regulated the expression of unique gene sets that were enriched for cell cycle, apoptosis, MAPK and insulin signaling compared to individual agents. To assess the potential efficacy of the combinatorial approach in human disease, we have developed a unique approach to study human prostate cancer where tumor specimens collected from men undergoing radical prostatectomy are cultured as explants for up to 7 days with maintenance of tissue integrity, cell proliferation and androgen signaling. Using this ex vivo strategy, we demonstrated that human prostate tumor tissue responds to the novel synthetic Hsp90 inhibitors (HSP990 and AUY922) with a marked reduction in proliferation and AR levels. Importantly, these studies have revealed a dissociation between client protein modulation (i.e. the established clinical biomarker of Hsp90 inhibition, HSP70) and proliferative response to Hsp90 inhibition, which was not seen in prostate cancer cell lines in vitro. Collectively, these findings suggest that the ex vivo methodology may better predict individual patient response to Hsp90 inhibitors and could be useful as a pre-clinical model of drug development, including identification of robust biomarkers of response. Currently we are using human tumor explants to identify optimal combinations of AR-targeting agents and potential markers of response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr CN08-05.

Herpesvirus saimiri-mediated delivery of the adenomatous polyposis coli tumour suppressor gene reduces proliferation of colorectal cancer cells

Colorectal cancer (CRC) is a major cause of cancer-related mortality. A contributing factor to the progression of this disease is sporadic or hereditary mutation of the adenomatous polyposis coli (APC) gene, a negative regulator of the Wnt signalling pathway. Inherited mutations in APC cause the disorder familial adenomatous polyposis (FAP), which leads to CRC development in early adulthood. However, the gene is also disrupted in some 60% of sporadic cancers. Restoration of functional APC may slow the growth of CRC by negatively regulating proliferation-associated genes such as c-myc. Therefore, we have cloned the cDNA of the APC tumour suppressor gene into a replication competent Herpesvirus saimiri (HVS)-based vector to assess APC gene delivery in SW480 and SW620 CRC cell lines. Our results demonstrate that full length APC protein was efficiently expressed from the HVS vector and that transgene expression inhibited proliferation of both the SW480 and the metastatic SW620 cancer cell lines. Moreover, a sustained effect could be observed for at least 8 weeks after initial infection in SW480 cells. In addition, monolayer wounding assays showed a marked reduction in proliferation and migration in HVS-GFP-APC infected cells. We believe that this is the first instance of infectious delivery and APC cDNA expression from a virus-based vector.

Assessment of the In vivo Antitumor Effects of ENMD-2076, a Novel Multitargeted Kinase Inhibitor, against Primary and Cell Line–Derived Human Colorectal Cancer Xenograft Models

This in vivo study was designed to investigate the efficacy of ENMD-2076, a small-molecule kinase inhibitor with activity against the Aurora kinases A and B, and several other tyrosine kinases linked to cancer, including vascular endothelial growth factor receptor 2, cKit, and fibroblast growth factor receptor 1, against murine xenograft models of human colorectal cancer (CRC). HT-29 CRC cell line xenografts were treated with either vehicle or ENMD-2076 (100 or 200 mg/kg) orally daily for 28 days. Tumor growth inhibition, dynamic contrast-enhanced magnetic resonance imaging, and (18)FDG-positron emission tomography were conducted to assess the antiproliferative, antiangiogenic, and antimetabolic responses, respectively. Effects on proliferation were also analyzed by immunohistochemical methods. Additionally, three patient-derived xenografts from primary and metastatic sites were treated with ENMD-2076 (100 mg/kg) and assessed for tumor growth inhibition. In the HT-29 xenograft model, ENMD-2076 induced initial tumor growth inhibition followed by regression. Treatment was associated with significant tumor blanching, indicating a loss of vascularity and substantial reductions in tumor vascular permeability and perfusion as measured by dynamic contrast-enhanced magnetic resonance imaging. Positron emission tomography scanning showed significant decreases in (18)FDG uptake at days 3 and 21 of treatment, which was associated with a marked reduction in proliferation as assessed by Ki-67. All three of the patient-derived xenografts tested were sensitive to treatment with ENMD 2076 as measured by tumor growth inhibition. ENMD-2076 showed robust antitumor activity against cell line and patient-derived xenograft models of CRC that is detectable by functional imaging, supporting clinical investigation of this agent in CRC.

ΔNp63α Repression of the Notch1 Gene Supports the Proliferative Capacity of Normal Human Keratinocytes and Cervical Cancer Cells

The p53 family member p63 is a master regulator of epithelial development. One of its isoforms, DeltaNp63alpha, is predominantly expressed in the basal cells of stratified epithelia and plays a fundamental role in control of regenerative potential and epithelial integrity. In contrast to p53, p63 is rarely mutated in human cancers, but it is frequently overexpressed in squamous cell carcinomas (SCC). However, its functional relevance to tumorigenesis remains largely unclear. We previously identified the Notch1 gene as a novel transcriptional target of p53. Here, we show that DeltaNp63alpha functions as a transcriptional repressor of the Notch1 gene through the p53-responsive element. Knockdown of p63 caused upregulation of Notch1 expression and marked reduction in proliferation and clonogenicity of both normal human keratinocytes and cervical cancer cell lines overexpressing DeltaNp63alpha. Concomitant silencing of Notch1 significantly rescued this phenotype, indicating the growth defect induced by p63 deficiency to be, at least in part, attributable to Notch1 function. Conversely, overexpression of DeltaNp63alpha decreased basal levels of Notch1, increased proliferative potential of normal human keratinocytes, and inhibited both p53-dependent and p53-independent induction of Notch1 and differentiation markers upon genotoxic stress and serum exposure, respectively. These results suggest that DeltaNp63alpha maintains the self-renewing capacity of normal human keratinocytes and cervical cancer cells partly through transcriptional repression of the Notch1 gene and imply a novel pathogenetical significance of frequently observed overexpression of DeltaNp63alpha together with p53 inactivation in SCCs.

Mechanisms of nicotine-induced resistance to treatment with chemotherapy and radiotherapy

e22008 Background: Nicotine (NIC), used for smoking cessation in cancer patients, is associated with increased tumor growth, angiogenesis, migration, invasion, growth factor production, and inhibition of apoptosis. The effects of NIC on the efficacy of radiotherapy (XRT) and/or chemotherapy (CT) have not been well characterized. Methods: A review of the literature produced a construct demonstrating that NIC generates more than 100 interactions with 40 proteins involved in tumor promotion and progression. From this construct, nicotinic acetylcholine receptors (nAChR), PI3K/Akt, and MAPK were identified as potential critical upstream targets modulating the effects of NIC. The effect of NIC on cell proliferation, cell survival, and protein activation was analyzed following CT, XRT, or chemoradiotherapy (CRT) in FaDu, A549, and H460 human cancer cell lines. Results: Proliferation measured with the WST-1 (tetrazolium) assay demonstrated that NIC increased proliferation following cisplatin, XRT, and cisplatin with XRT. NIC administration increased cell survival (colony survival) following XRT and following CT (cisplatin, doxorubicin, or etoposide) regimens in all cell lines. nAChR expression was verified in each cell line using Western blot. Non-specific inhibition of nAChR demonstrated no consistent effect on preventing NIC induced resistance to CT, XRT, or CRT; however, specific inhibition of the alpha-3- or alpha-7-nAChR had variable cell specific ability to prevent NIC induced resistance. Inhibition of MAPK activity (using PD98059) produced marked reduction in proliferation of all cell lines, but generally had no effect on NIC induced resistance. PI3K inhibition (using LY294002) generally increased sensitivity to cisplatin and XRT; however, some PI3K activity was required to achieve the full effect of cisplatin. NIC prevented LY294002 induced sensitization to chemotherapy with no effect on sensitization to XRT. Conclusions: Nicotine induces a broad spectrum of resistance to CT, XRT, and CRT and serves as an excellent model of treatment resistance. Mechanisms of NIC induced resistance to CT and XRT appear to diverge. These findings have significant clinical implications including smoking cessation, treatment efficacy, and outcomes research in cancer patients. No significant financial relationships to disclose.

Radiation-Free Anti-CD3-Conditioning Regimen Maintains Tissue Protection Mechanisms and Prevents GVHD: Role of Tissue Expression of B7H1

We reported that donor CD8+ T cells mediated graft versus leukemia (GVL) activity without causing graft versus host disease (GVHD) in anti-CD3-conditioned recipients, although the same dose of donor CD8+ T cells caused lethal GVHD in recipients conditioned with sublethal total body irradiation (TBI) (J. Immunol. 2007). We recently observed that donor CD8+ T cells from the liver of anti-CD3-conditioned recipients showed a marked reduction in proliferation in response to anti-CD3/CD28 stimulation, as compared to those from TBI-conditioned recipients, indicating that donor CD8+ T cells are tolerized in the tissues of anti-CD3-conditioned recipients. B7H1, a co-inhibitory molecule, is constitutively expressed by hematopoietic cells and expressed by parenchymal cells after IFN-γ induction. B7H1 tolerizes T cells by interaction with its ligand PD-1 on activated T cells. To explore the role of B7H1 in GVHD prevention in anti-CD3-conditioned recipients, donor C57BL/6 CD8+ T cells (20×106) and bone marrow cells (100×106) were transplanted into anti-CD3-conditioned wild-type or B7H1−/− BALB/c recipients. While the wild-type recipients all survived for more than 100 days without signs of GVHD, the B7H1−/− recipients developed severe GVHD with diarrhea, weight-loss, and hair-loss, and 70% of them died 60 days after transplantation. Similarly, while donor CD8+ T and BM cells induced little GVHD in unconditioned Rag-2−/− BALB/c recipients, they induced severe lethal GVHD in B7H1−/− Rag-2−/− BALB/c recipients. Furthermore, donor CD8+ T and BM cells still induced lethal GVHD in unconditioned chimeric B7H1−/−Rag-2−/− recipients reconstituted with B7H1+/+Rag-2−/− BM. In addition, we observed upregulation of B7H1 expression by hepatocytes and intestine epithelial cells of anti-CD3-conditioned BALB/c or unconditioned Rag-2−/− recipients after donor cells infusion, as judged by RT-PCR, flow cytometry analysis, and histoimmunostaining of B7H1. In vivo bioluminescent imagine showed much more severe tissue infiltration of donor T cells in B7H1−/− recipients as compared to B7H1+/+ recipients, and the in vitro proliferation of donor CD8+ T cells from the liver of the former recipients was much more vigorous than that of the latter recipients. These results demonstrate that B7H1 expression by tissue parenchymal cells rather than hematopoietic cells tolerizes infiltrating donor T cells in GVHD target tissues and prevents GVHD; and that the radiation-free anti-CD3-conditioning regimen can maintain this tissue protection mechanism.(This study is surpported by Marcus Foundation and NIH R01 AI 066008).

Enhanced BCR-ABL kinase inhibition does not result in increased inhibition of downstream signaling pathways or increased growth suppression in CML progenitors

The therapeutic success of imatinib in chronic myeloid leukemia (CML) is hampered by persistence of malignant stem cells. We investigated whether nilotinib, a more potent BCR-ABL kinase inhibitor could target CML primitive progenitors more effectively than imatinib. CML and normal progenitor cells were cultured with nilotinib or imatinib in growth factor supplemented medium. Nilotinib inhibited BCR-ABL kinase activity at lower concentrations than imatinib. Nilotinib inhibited mitogen-activated protein kinase (MAPK), AKT and STAT5 phosphorylation in CML CD34(+) cells in the absence of growth factors (GFs), but did not suppress AKT and STAT5 activity, and resulted in increased MAPK activity, in the presence of GFs. Nilotinib and imatinib resulted in similar suppression of CML primitive and committed progenitors in long-term culture-initiating cell and colony-forming cell assays. Inhibition of progenitor growth was related to marked reduction in proliferation, but only a modest increase in apoptosis. Nilotinib did not show increased efficacy in reducing nondividing CML progenitors compared with imatinib. These results indicate that more potent tyrosine kinase inhibitors by themselves will not be more effective in eliminating CML progenitors than imatinib and that additional mechanism required for maintenance of malignant stem cells need to be identified to improve targeting of leukemia stem cells.