Richard Ottman

MicroRNA expressions associated with progression of prostate cancer cells to antiandrogen therapy resistance

BackgroundDevelopment of resistance to androgen deprivation therapy (ADT) is a major obstacle for the management of advanced prostate cancer. Therapies with androgen receptor (AR) antagonists and androgen withdrawal initially regress tumors but development of compensatory mechanisms including AR bypass signaling leads to re-growth of tumors. MicroRNAs (miRNAs) are small regulatory RNAs that are involved in maintenance of cell homeostasis but are often altered in tumor cells.ResultsIn this study, we determined the association of genome wide miRNA expression (1113 unique miRNAs) with development of resistance to ADT. We used androgen sensitive prostate cancer cells that progressed to ADT and AR antagonist Casodex (CDX) resistance upon androgen withdrawal and treatment with CDX. Validation of expression of a subset of 100 miRNAs led to identification of 43 miRNAs that are significantly altered during progression of cells to treatment resistance. We also show a correlation of altered expression of 10 proteins targeted by some of these miRNAs in these cells.ConclusionsWe conclude that dynamic alterations in miRNA expression occur early on during androgen deprivation therapy, and androgen receptor blockade. The cumulative effect of these altered miRNA expression profiles is the temporal modulation of multiple signaling pathways promoting survival and acquisition of resistance. These early events are driving the transition to castration resistance and cannot be studied in already developed CRPC cell lines or tissues. Furthermore our results can be used a prognostic marker of cancers with a potential to be resistant to ADT.

LIM kinase1 modulates function of membrane type matrix metalloproteinase 1: implication in invasion of prostate cancer cells

BackgroundLIM kinase 1 (LIMK1) is an actin and microtubule cytoskeleton modulatory protein that is overexpressed in a number of cancerous tissues and cells and also promotes invasion and metastasis of prostate and breast cancer cells. Membrane type matrix metalloproteinase 1 (MT1-MMP) is a critical modulator of extracellular matrix (ECM) turnover through pericellular proteolysis and thus plays crucial roles in neoplastic cell invasion and metastasis. MT1-MMP and its substrates pro-MMP-2 and pro-MMP-9 are often overexpressed in a variety of cancers including prostate cancer and the expression levels correlate with the grade of malignancy in prostate cancer cells. The purpose of this study is to determine any functional relation between LIMK1 and MT1-MMP and its implication in cell invasion.ResultsOur results showed that treatment with the hydroxamate inhibitor of MT1-MMP, MMP-2 and MMP-9 ilomastat inhibited LIMK1-induced invasion of benign prostate epithelial cells. Over expression of LIMK1 resulted in increased collagenolytic activity of MMP-2, and secretion of pro-MMP2 and pro-MMP-9. Cells over expressing LIMK1 also exhibited increased expression of MT1-MMP, transcriptional activation and its localization to the plasma membrane. LIMK1 physically associates with MT1-MMP and is colocalized with it to the Golgi vesicles. We also noted increased expression of both MT1-MMP and LIMK1 in prostate tumor tissues.ConclusionOur results provide new information on regulation of MT1-MMP function by LIMK1 and showed for the first time, involvement of MMPs in LIMK1 induced cell invasion.

In-fiber production of polymeric particles for biosensing and encapsulation

Significance A scalable, chemistry-independent, fluid-instability–mediated in-fiber route for fabricating uniformly sized spherical polymeric particles over a wide span of diameters is developed targeting biomedical applications. Both surface functionalization of solid biocompatible polymer particles for protein–protein interactions and volume encapsulation of a biological material in spherical hollow polymer shells are confirmed, in addition to combining both surface and volumetric functionalities in the same polymeric particle. Polymeric micro- and nanoparticles are becoming a mainstay in biomedicine, medical diagnostics, and therapeutics, where they are used in implementing sensing mechanisms, as imaging contrast agents, and in drug delivery. Current approaches to the fabrication of such particles are typically finely tuned to specific monomer or polymer species, size ranges, and structures. We present a general scalable methodology for fabricating uniformly sized spherical polymeric particles from a wide range of polymers produced with complex internal architectures and continuously tunable diameters extending from the millimeter scale down to 50 nm. Controllable access to such a wide range of sizes enables broad applications in cancer treatment, immunology, and vaccines. Our approach harnesses thermally induced, predictable fluid instabilities in composite core/cladding polymer fibers drawn from a macroscopic scaled-up model called a “preform.” Through a stack-and-draw process, we produce fibers containing a multiplicity of identical cylindrical cores made of the polymers of choice embedded in a polymer cladding. The instability leads to the breakup of the initially intact cores, independent of the polymer chemistry, into necklaces of spherical particles held in isolation within the cladding matrix along the entire fiber length. We demonstrate here surface functionalization of the extracted particles for biodetection through specific protein–protein interactions, volumetric encapsulation of a biomaterial in spherical polymeric shells, and the combination of both surface and volumetric functionalities in the same particle. These particles used in distinct modalities may be produced from the desired biocompatible polymer by changing only the geometry of the macroscopic preform from which the fiber is drawn.

A functional cooperativity between Aurora A kinase and LIM kinase1: Implication in the mitotic process

Aurora kinase A (Aur-A), a mitotic kinase, regulates initiation of mitosis through centrosome separation and proper assembly of bipolar spindles. LIM kinase 1 (LIMK1), a modulator of actin and microtubule dynamics, is involved in the mitotic process through inactivating phosphorylation of cofilin. Phosphorylated LIMK1 is recruited to the centrosomes during early prophase, where it colocalizes with γ-tubulin. Here, we report a novel functional cooperativity between Aur-A and LIMK1 through mutual phosphorylation. LIMK1 is recruited to the centrosomes during early prophase and then to the spindle poles, where it colocalizes with Aur-A. Aur-A physically associates with LIMK1 and activates it through phosphorylation, which is important for its centrosomal and spindle pole localization. Aur-A also acts as a substrate of LIMK1, and the function of LIMK1 is important for its specific localization and regulation of spindle morphology. Taken together, the novel molecular interaction between these two kinases and their regulatory roles on one another’s function may provide new insight on the role of Aur-A in manipulation of actin and microtubular structures during spindle formation.

The other face of miR-17-92a cluster, exhibiting tumor suppressor effects in prostate cancer

miR-17-92a cluster miRNAs are transcribed from a polycistronic transcription unit C13orf25 that generates six mature miRNAs, miR-17, miR-18a, miR-19a, miR-19b, miR-20a and miR-92a that are overexpressed in lung and colon cancers. Here we show that the expression of miR-17-92a miRNAs are reduced in cancerous prostate tissues compared to uninvolved areas and also in aggressive prostate cancer cells. Restoration of expression of all members of miR-17-92a cluster showed, decreased expression of cell cycle regulatory proteins cyclin D1 and SSH1; and LIMK1 and FGD4 of RhoGTPase signaling pathway. Expression of miR-17-92a miRNAs caused decreased cell proliferation, reduced activation of AKT and MAP kinases, delayed tumorigenicity and reduced tumor growth in animals. Expression of miR-17-92a miRNAs inhibited EMT via reduced cell migration and expression of mesenchymal markers while elevating expression and surface localization of the epithelial marker E-Cadherin. Expression of miR-17-92a miRNAs improved sensitivity of androgen dependent LNCaP 104-S prostate cancer cells to anti-androgen drug Casodex, AKT inhibitor MK-2206 2HCl, and docetaxel. The androgen refractory PC-3 cells also showed increased sensitivity to docetaxel, MK-2206 2HCl and Aurora kinase inhibitor VX680 upon ectopic expression of miR-17-92a cluster miRNAs. Our data demonstrate a tumor suppressor effect of miR-17-92a cluster miRNAs in prostate cancer cells and restoration of expression of these miRNAs has a therapeutic benefit for both androgen-dependent and -independent prostate cancer cells.

Abstract LB-325: Tumor suppressor effects of miR-17-92a cluster miRNAs on prostate cancer

MicroRNAs in miR-17-92a cluster are transcribed from a polycistronic transcription unit C13orf25 that generates six mature miRNAs, miR-17, miR-18a, miR-19a, miR- 19b, miR-20a. These miRNAs are reported to be overexpressed in colon and lung cancers, however, we noted a loss of expression of all members of this miR cluster in prostate cancer tissues compared to uninvolved tissues and in aggressive prostate cancer cells. In this study, we show that replenishment of miR-17-92a cluster as a whole showed antiproliferative effects, reduced activation of AKT and MAP kinases, delayed tumorigenicity and reduced tumor growth. Analysis of the expression of mRNA targets showed reduced expression of cell cycle regulatory proteins cyclin D1 and SSH1; and LIMK1 and FGD4 of RhoGTPase signaling pathway through direct targeting. miR-17-92 cluster expression also inhibited EMT via reduced cell migration and expression of mesenchymal markers while elevating expression and surface localization of the epithelial marker E-Cadherin. miR-17-92a miRNAs improved sensitivity of androgen dependent LNCaP 104-S prostate cancer cells to anti-androgen drug Casodex, AKT inhibitor MK-2206 2HCl, and docetaxel. The androgen refractory PC-3 cells also showed increased sensitivity to docetaxel, MK-2206 2HCl and Aurora kinase inhibitor VX680 upon ectopic expression of miR-17-92a cluster miRNAs. RNA seq analysis of prostate cancer cells expressing miR-17-92a cluster revealed altered expression of a number of oncogenic and tumor suppressor mRNAs, and noncoding RNAs, which may have implications in the altered phenotype of these cells. Our data demonstrate a tumor suppressor effect of miR-17-92a cluster miRNAs in prostate cancer cells and restoration of expression of these miRNAs has a therapeutic benefit for both androgen-dependent and -independent prostate cancer cells. Note: This abstract was not presented at the meeting. Citation Format: Ratna Chakrabarti, Richard Ottman, Md Faqrul Hasan. Tumor suppressor effects of miR-17-92a cluster miRNAs on prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-325. doi:10.1158/1538-7445.AM2017-LB-325

Scalable Production of Digitally Designed Multifunctional Polymeric Particles by In-Fiber Fluid Instabilities

By exploiting fluid instabilities in multimaterial fibers, we present a fabrication methodology for producing multifunctional particles. Particles are produced with optical and magnetic dopants confined to specific compartments within the particle.

Abstract 1171: LIMK1 functions as a modulator of expression and targeting of CXCR4 in prostate cancer cells through a negative feedback loop

Increased activation of the GPCR CXCR4/CXCL12 signaling axis is commonly noted in metastatic prostate cancer and shows an association with poor prognosis. CXCR4, and its ligand, CXCL12/SDF-1a, are recognized as powerful mediators for tumor microenvironment remodeling and chemoprotection. LIM kinase 1 (LIMK1), a serine/threonine kinase, is an actin and microtubule modulatory protein that is also overexpressed in a variety of cancers including prostate cancer. Earlier, we showed that LIMK1 is involved in cell invasion and inhibition of LIMK1 expression reverted the invasive phenotype of PC3 prostate cancer cells. Activation of CXCR4/CXCL12 axis promotes activation of LIMK1 through RhoGTPAse pathway and promotes docetaxel resistance. Here we show that LIMK1 expression and functions have a positive correlation with CXCR4 expression and subcellular localization. We used metastatic prostate cancer cell lines PC3 and M12 and the non-tumorigenic prostate cell lines BPH and P69 to monitor expression and membrane localization of CXCR4 using western blots, flow cytometry and immunofluorescence analysis. Our analysis showed that cells with higher expression of LIMK1 (PC3) overexpress CXCR4, and that cell lines with low levels of LIMK1 (BPH and P69) express relatively lower amounts of CXCR4. Flow cytometric analysis showed a stronger surface staining of CXCR4 in the LIMK1 overexpressing PC3 cells compared to BPH cells. Next, we studied the effect of shRNA-mediated inhibition of LIMK1 expression on CXCR4 expression in PC3 or M12 cells. Western blot and immunofluorescence analysis showed noticeable reduction in CXCR4 levels, and its surface localization, as noted by flow cytometry, in LIMK1 shRNA transfected PC3 and M12 cells. To assess if the kinase activity of LIMK1 is essential for CXCR4 expression and surface localization, we treated these cells with LIMK1 specific kinase inhibitor BMS-5 and determined expression of CXCR4. Western blots and immunofluorescence analysis showed reduced expression of CXCR4 in treated cells compared to vehicle treated cells. Our observation indicates an indirect association between LIMK1 and CXCR4 expression and shows for the first time that expression of LIMK1 is required for the maintenance of CXCR4 expression. Our study demonstrates a novel positive feed back regulatory mechanism between LIMK1 and CXCR4 and suggest a possible combination treatment option through inhibition of LIMK1 expression and/or its kinase activity for improving chemosensitivity of prostate cancer cells with overactive CXCR4/CXCL12 axis. Citation Format: Richard Ottman, Lisa Ritchey, Ryan Herchan, Alexia Bossan, Ratna Chakrabarti. LIMK1 functions as a modulator of expression and targeting of CXCR4 in prostate cancer cells through a negative feedback loop. [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 1171.

Abstract 5221: Association of miR-17-92 cluster and its target, Frabin, with development of aggressive prostate cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Androgen blockade therapy has become the mainstay for advanced prostate cancer. However, prolonged androgen blockade leads to outgrowth of androgen independent (AI) cells and the development of castration resistant prostate cancer (CRPC). The transition to androgen independence can occur through several adaptive mechanisms and usually results in the acquisition of a more aggressive phenotype, compared to their androgen sensitive progenitors. Recently, the role of MicroRNAs (miRNAs) has been demonstrated in regulation of gene expression for cancer progression, metastasis, and resistance to therapeutic strategies. However, the role of miRNAs in progression of androgen sensitive prostate cancer to CRPC has not been clearly defined. To study this transition, we subjected androgen sensitive (AS) LNCaP prostate cancer cells to androgen deprivation and androgen receptor antagonist bicalutamide (CDX) therapy until a subset of cells (CDXR) survived. Genome-wide expression profiling of miRNAs identified a subset of miRNAs that are significantly deregulated in these cells. The miR-17-92 cluster is one of the groups of miRNAs that becomes down regulated as the cancer cells progresses towards androgen blockade therapy (ADT) resistance. More than a 4 to 24-fold down regulation of these miRNAs were noted in CDXR compared to AS LNCaP cells. We monitored the expression status of the miR-17-92 cluster in patient tumor tissues, which showed down regulation of these miRNAs in 64-82% of the tissues tested. Target prediction database searches identified FGD4/Frabin, a novel RhoGEF as one of the targets of this cluster. Previous studies have shown Frabin to be involved in filopodia formation and cell migration through interaction with CDC42. Beyond this, little is known about Frabin function in cancer or whether it is involved in the development of CRPC. Western blot analysis of treated cell lysates confirmed increased expression of Frabin in AI and CDXR LNCaP cells. Analysis in tissue microarray (267 cores) showed a significant up regulation of Frabin in advanced prostate cancer tissues including AI specimens. More than 90% of the AI tissues and 88% of tissues with 8-10 Gleason scores showed a median staining intensity between 2-3-fold higher compared to BPH tissues. Ectopic expression of mir-17-92 clusters in AI PC3 cells down regulated Frabin expression and improved sensitivity of these cells to docetaxel (DTX) treatments. Our results show a novel involvement of a miRNA cluster/mRNA axis in development of AI and aggressive prostate cancer. This study also provides important insight into the molecular mechanism of development of CRPC and identifies potential biomarkers and therapeutic targets for management of advanced prostate cancer. Citation Format: Richard James Ottman, Jenna Levy, Ratna Chakrabarti. Association of miR-17-92 cluster and its target, Frabin, with development of aggressive prostate cancer. [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 5221. doi:10.1158/1538-7445.AM2014-5221

Abstract 1950: Signature MicroRNAs involved In transition of androgen sensitive prostate cancer cells to androgen-insensitive ones during androgen deprivation therapy.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Because of the clinical efficacy, androgen blockade therapy has become the mainstay for advanced prostate cancer. However, prolonged androgen blockade either by androgen deprivation or by the use of androgen receptor antagonists, leads to activation of various adaptive mechanisms, which promotes development of hormone-refractory prostate cancer (HRPC). Microarray analysis revealed an increasingly complex profile of gene expression in prostate cancer with respect to the status of androgen sensitivity or refractoriness but the process of progression to androgen insensitivity is not well understood. Recently, role of MicroRNAs (miRNA) has been demonstrated in regulation of gene expression for cancer progression. Aberrant expression of miRNAs has been correlated with tumor differentiation, metastasis, and resistance to therapeutic strategies. However, the role of miRNAs in progression of androgen sensitive prostate cancer to HRPC has not been clearly defined. In this study, we examined changes in the expression profiles of miRNAs at the genome level during development of HRPC. Androgen sensitive LNCaP prostate cancer cells were subjected to androgen deprivation until a subset of cells remained, which survived in the absence of androgen. Samples were collected at different time points along the treatment and expression of 1,113 miRNAs were monitored through qRT-PCR. Initial analysis identified 140 miRNas that had more than three-fold reduction in expression and 137 miRNAs that had more than three-fold elevated expression compared to untreated samples. Of these, 100 miRNAs were selected for further validation, and finally 41 miRNAs were chosen that displayed the most significant changes with treatment. Using online tools and databases we determined potential mRNA targets of these miRNAs based on sequence homology. MRNA targets with the highest binding scores and especially those that were targeted by multiple miRNAs were further investigated. We found many of these putatively down regulated mRNAs code for transcriptional regulators or were involved in pro-apoptotic signaling. In contrast many of the putatively up regulated mRNAs code for oncoproteins and proteins involved in mitogenic signaling. Western blot analysis confirmed that the expression profiles of these proteins are indeed in correlation with the expression profiles of the miRNAs identified. Our results provide important insight into the development of HRPC and additionally, identify potential biomarkers and therapeutic targets for management of advanced prostate cancer. Citation Format: Richard J. Ottman, Robert Lorch, Camha Nguyen, Ratna Chakrabarti. Signature MicroRNAs involved In transition of androgen sensitive prostate cancer cells to androgen-insensitive ones during androgen deprivation therapy. [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 1950. doi:10.1158/1538-7445.AM2013-1950