Loukia G. Karacosta

A Regulatory Feedback Loop Between Ca2+/Calmodulin-dependent Protein Kinase Kinase 2 (CaMKK2) and the Androgen Receptor in Prostate Cancer Progression

Background: Defining molecular mechanisms that regulate AR activity is critical for understanding prostate cancer progression. Results: CaMKK2 increases during disease progression, is transcriptionally regulated by the AR, promotes proliferation, and is required for optimal AR transcriptional activity. Conclusion: CaMKK2 is in a feedback circuit to maintain AR activity. Significance: The CaMKK2 pathway is a promising target for prostate cancer therapy. The androgen receptor (AR) plays a critical role in prostate cancer (PCa) progression, however, the molecular mechanisms by which the AR regulates cell proliferation in androgen-dependent and castration-resistant PCa are incompletely understood. We report that Ca2+/calmodulin-dependent kinase kinase 2 (CaMKK2) expression increases and becomes nuclear or perinuclear in advanced PCa. In the TRAMP (transgenic adenocarcinoma of mouse prostate) model of PCa, CaMKK2 expression increases with PCa progression with many cells exhibiting nuclear staining. CaMKK2 expression is higher in human castration-resistant tumor xenografts compared with androgen-responsive xenografts and is markedly higher in the AR-expressing, tumorigenic cell line LNCaP compared with cell lines that are AR-nonexpressing and/or nontumorigenic. In LNCaP cells, dihydrotestosterone induced CaMKK2 mRNA and protein expression and translocation of CaMKK2 to the nucleus. Conversely, androgen withdrawal suppressed CaMKK2 expression. Knockdown of CaMKK2 expression by RNAi reduced LNCaP cell proliferation and increased percentages of cells in G1 phase, whereas correspondingly reducing percentages in S phase, of the cell cycle. CaMKK2 knockdown reduced expression of the AR target gene prostate-specific antigen at both mRNA and protein levels, AR transcriptional activity driven by androgen responsive elements from the prostate-specific probasin gene promoter and levels of the AR-regulated cell cycle proteins, cyclin D1 and hyperphosphorylated Rb. Our results suggest that in PCa progression, CaMKK2 and the AR are in a feedback loop in which CaMKK2 is induced by the AR to maintain AR activity, AR-dependent cell cycle control, and continued cell proliferation.

Nucleoporin 62 and Ca(2+)/calmodulin dependent kinase kinase 2 regulate androgen receptor activity in castrate resistant prostate cancer cells.

Re‐activation of the transcriptional activity of the androgen receptor (AR) is an important factor mediating progression from androgen‐responsive to castrate‐resistant prostate cancer (CRPC). However, the mechanisms regulating AR activity in CRPC remain incompletely understood. Ca2+/calmodulin‐dependent kinase kinase (CaMKK) 2 was previously shown to regulate AR activity in androgen‐responsive prostate cancer cells. Our objective was to further explore the basis of this regulation in CRPC cells.

Akt Activation by Ca2+/calmodulin-dependent protein Kinase Kinase 2 (CaMKK2) in Ovarian Cancer Cells

Hyperactivation of Akt is associated with oncogenic changes in the growth, survival, and chemoresistance of cancer cells. The PI3K/phosphoinositide-dependent kinase (PDK) 1 pathway represents the canonical mechanism for phosphorylation of Akt at its primary activation site, Thr-308. We observed that Ca2+/calmodulin (CaM)-dependent protein kinase kinase 2 (β) (CaMKK2) is highly expressed in high-grade serous ovarian cancer, and we investigated its role in Akt activation in ovarian cancer (OVCa) cell lines (OVCAR-3, SKOV-3, and Caov-3). Knockdown or pharmacological inhibition of CaMKK2 produced phenotypes expected of Akt inhibition, including reductions in cell growth and cell viability and in the regulation of Akt downstream targets involved in G1/S transition and apoptosis. CaMKK2 knockdown or inhibition decreased Akt phosphorylation at Thr-308 and Ser-473 to extents similar to those of PDK1 knockdown or PI3K inhibition. Combined CaMKK2 and PDK1 knockdown or CaMKK and PI3K inhibition, respectively, produced additive effects on p-Akt and cell growth, consistent with direct Akt phosphorylation by CaMKK2. This conclusion was supported by the absence of effects of CaMKK2 knockdown/inhibition on alternative means of activating Akt via p-Akt Thr-450, p-PDK1 Ser-241, or p-IRS1 Ser-636/639. Recombinant CaMKK2 directly activated recombinant Akt by phosphorylation at Thr-308 in a Ca2+/CaM-dependent manner. In OVCa cells, p-Akt Thr-308 was significantly inhibited by intracellular Ca2+i chelation or CaM inhibition. Ionomycin-induced Ca2+ influx promoted p-Akt, an effect blocked by PDK1, and/or CaMKK2, siRNAs, and by PI3K and/or CaMKK inhibitors. CaMKK2 knockdown potentiated the effects of the chemotherapeutic drugs carboplatin and PX-866 to reduce proliferation and survival of OVCa cells.

Humanization of JAA-F11, a Highly Specific Anti-Thomsen-Friedenreich Pancarcinoma Antibody and In Vitro Efficacy Analysis

JAA-F11 is a highly specific mouse monoclonal to the Thomsen-Friedenreich Antigen (TF-Ag) which is an alpha-O-linked disaccharide antigen on the surface of ~80% of human carcinomas, including breast, lung, colon, bladder, ovarian, and prostate cancers, and is cryptic on normal cells. JAA-F11 has potential, when humanized, for cancer immunotherapy for multiple cancer types. Humanization of JAA-F11, was performed utilizing complementarity determining regions grafting on a homology framework. The objective herein is to test the specificity, affinity and biology efficacy of the humanized JAA-F11 (hJAA-F11). Using a 609 target glycan array, 2 hJAA-F11 constructs were shown to have excellent chemical specificity, binding only to TF-Ag alpha-linked structures and not to TF-Ag beta-linked structures. The relative affinity of these hJAA-F11 constructs for TF-Ag was improved over the mouse antibody, while T20 scoring predicted low clinical immunogenicity. The hJAA-F11 constructs produced antibody-dependent cellular cytotoxicity in breast and lung tumor lines shown to express TF-Ag by flow cytometry. Internalization of hJAA-F11 into cancer cells was also shown using a surface binding ELISA and confirmed by immunofluorescence microscopy. Both the naked hJAA-F11 and a maytansine-conjugated antibody (hJAA-F11-DM1) suppressed in vivo tumor progression in a human breast cancer xenograft model in SCID mice. Together, our results support the conclusion that the humanized antibody to the TF-Ag has potential as an adjunct therapy, either directly or as part of an antibody drug conjugate, to treat breast cancer, including triple negative breast cancer which currently has no targeted therapy, as well as lung cancer.

Preclinical Analysis of JAA-F11, a Specific Anti-Thomsen-Friedenreich Antibody via Immunohistochemistry and In Vivo Imaging.

The tumor specificity of JAA-F11, a novel monoclonal antibody specific for the Thomsen-Friedenreich cancer antigen (TF-Ag-alpha linked), has been comprehensively studied by in vitro immunohistochemical (IHC) staining of human tumor and normal tissue microarrays and in vivo biodistribution and imaging by micro-positron emission tomography imaging in breast and lung tumor models in mice. The IHC analysis detailed herein is the comprehensive biological analysis of the tumor specificity of JAA-F11 antibody performed as JAA-F11 is progressing towards preclinical safety testing and clinical trials. Wide tumor reactivity of JAA-F11, relative to the matched mouse IgG3 (control), was observed in 85% of 1269 cases of breast, lung, prostate, colon, bladder, and ovarian cancer. Staining on tissues from breast cancer cases was similar regardless of hormonal or Her2 status, and this is particularly important in finding a target on the currently untargetable triple-negative breast cancer subtype. Humanization of JAA-F11 was recently carried out as explained in a companion paper “Humanization of JAA-F11, a Highly Specific Anti–Thomsen-Friedenreich Pancarcinoma Antibody and In Vitro Efficacy Analysis” (Neoplasia 19: 716-733, 2017), and it was confirmed that humanization did not affect chemical specificity. IHC studies with humanized JAA-F11 showed similar binding to human breast tumor tissues. In vivo imaging and biodistribution studies in a mouse syngeneic breast cancer model and in a mouse-human xenograft lung cancer model with humanized 124I- JAA-F11 construct confirmed in vitro tumor reactivity and specificity. In conclusion, the tumor reactivity of JAA-F11 supports the continued development of JAA-F11 as a targeted cancer therapeutic for multiple cancers, including those with unmet need.

Corrigendum to “Humanization of JAA-F11, a Highly Specific Anti-Thomsen-Friedenreich Pancarcinoma Antibody and In Vitro Efficacy Analysis” [Neoplasia 19.9 (2017) 716-733]

*Department of Biotechnical and Clinical Laboratory Sciences, University at Buffalo, Buffalo, NY; For-Robin, Inc., Buffalo, NY; Department of Microbiology and Immunology, University at Buffalo, Buffalo, NY; Research Service, Harry S. Truman Memorial Veterans Hospital, Columbia, MO; Department of Pathology and Anatomical Sciences, University of Missouri, Columbia, MO; Department of Medical Pharmacology and Physiology, University of Missouri, Columbia, MO; Goodwin Biotechnology, Inc., Plantation, FL; Department of Pharmacology and Toxicology, University at Buffalo, Buffalo, NY

Abstract 576: Immunohistochemistry and radioimaging with hJAA-11 antibody to the Thomsen-Friedenreich antigen: Potential theranostic application for breast cancer

Theranostics through the utilization of immunohistochemistry followed by radioimaging to determine if metastatic foci will react with a therapeutic antibody will allow for the selection of the patient population that will most benefit from this immunotherapy. The Thomsen-Friedenreich antigen (TF-Ag) has been shown to be involved in ∼90% of carcinomas, specifically breast carcinomas, making it a suitable target for radioimaging and therapy. The anti-TF-Ag antibody, JAA-F11, a mouse monoclonal antibody (mAb), has had success in localization, blocking metastasis, and inhibiting cell proliferation, and binds to ∼80% of breast cancer cell lines, without preference to receptor status. This is significant since the triple negative breast cancer (ER-/PR-/HER2-) has no current targeted treatment. Studies are extended to human breast cancer tissue microarray immunohistochemical (IHC) analysis, which was scored blindly by a pathologist on a semi- quantitative scale. JAA-F11 stained approximately 76% of all breast cancer specimens, which included cases of mucinous, medullary, invasive ductal and neuroendocrine carcinomas. The staining observed was irrespective of receptor status, whereas in normal breast tissue, staining was either absent or very weak/weak. In addition, all available matched lymph node metastasis stained, with greater intensity observed in 39% of cases. JAA-F11 IHC studies performed on human normal organ tissue arrays, showed staining that for the most part was not significantly different from staining obtained with isotype control antibody, or was observed in areas that would not be therapeutically accessible. Furthermore, in an additional IHC study, preliminary results suggest that JAA-F11 significantly stained other carcinomas including those of the colon, bladder, ovary and prostate. In vitro studies show that the humanized JAA-F11 (hJAA-F11) has similar chemical specificity and higher affinity towards the TF-Ag. Imaging studies were performed in a BALB/c mouse breast cancer model with the hJAA-F11 to determine biological reactivity and to predict the feasibility of theranostic imaging prior to therapy. After ∼10 days of tumor growth, mice pretreated with cold iodine water and rabbit IgG to inhibit binding to Fc receptors, were given tail vein injections of hJAA-F11 conjugated with [124]-I. Clear tumor images were obtained up to 144 hours post injection. Biodistribution analysis has provided further results indicating increased%ID/g (7.0±3.9%) in tumor tissue as compared to healthy tissues (brain%ID/g to be 0.21±.09, stomach 0.80±0.19%, and bone 0.90±2.4%). Results support that the hJAA-F11 antibody can be used in a multi-step theranostic approach, with analysis of tumor binding in IHC, followed by imaging to determine in vivo tumor targeting prior to direct immunotherapy or antibody drug conjugate therapy with hJAA-F11. Citation Format: Loukia Karacosta, Holly Johnson, Julia Abdullah, Taylor Chrisikos, Rachel Ludwig, Bradley Turner, Swetha Tati, Diala Ghazal, Munnawar Sajjad, Stephen Koury, Susan Morey, Julie Adams, Kate Rittenhouse-Olson. Immunohistochemistry and radioimaging with hJAA-11 antibody to the Thomsen-Friedenreich antigen: Potential theranostic application for breast cancer. [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 576.

Abstract B13: Regulation of Akt activity, cell proliferation, and viability in ovarian cancer cells by calcium/calmodulin-dependent protein kinase kinase 2

Calcium/Calmodulin-dependent protein kinase kinase 2 (CaMKK2) has been implicated in the regulation of G1/S phase cell cycle progression in prostate and breast cancer but has not been studied in ovarian cancer (OvCa). We report here the following. CaMKK2 is abundantly expressed in high grade serous papillary cystadenocarcinoma. Knockdown (KD) of CaMKK2 expression using RNAi in OVCAR-3 OvCa cells resulted in slowing of cell growth, decreased cyclin D1 protein and mRNA, decreased phosphorylation of the tumor suppressor protein Rb at Ser807/811 and decreased DNA synthesis, indicating an arrest at the G1/S interface. CaMKK2 KD decreased cell viability, increased PARP cleavage and increased Caspase-3/7 activity suggesting apoptosis induction by CaMKK2 loss. CaMKK2 KD decreased p-Akt at Thr308 to a similar extent (59.3%) as that of PDK1 KD (50.4%) in OVCAR-3 cells. Combined CaMKK2 and PDK1 KDs led to an additive (80.7%) decrease in p-Akt suggesting that CaMKK2 regulates Akt activating phosphorylation independently of the canonical-PDK1 pathway. Additive effects of combined PDK1/CaMKK2 KD on p-Akt Thr308 in SKOV-3 OvCa cells were also obtained. The ability of baculovirus expressed and purified Akt to phosphorylate a specific peptide substrate was enhanced at least 9-fold by purified CaMKK2, whereas kinase activity of Akt T308A/S473A was not increased by CaMKK2. Direct phosphorylation of baculovirus expressed Akt at Thr308 by CaMKK2 was confirmed by Western blotting using Akt phospho-specific antibodies. Altogether these data show that CaMKK2 regulates cell growth and viability in OvCa cells and directly phosphorylates Akt to promote its activity via a non-PDK1-dependent pathway. These data indicate that CaMKK2 may be a novel therapeutic target for OvCa in combination with a PI3K inhibitor. Citation Format: Angela M. Gocher, Thomas F. Franke, Gissou Azabdaftari, Loukia G. Karacosta, Arthur M. Edelman. Regulation of Akt activity, cell proliferation, and viability in ovarian cancer cells by calcium/calmodulin-dependent protein kinase kinase 2. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B13.

Abstract A10: CaMKK2 regulates cellular proliferation and androgen receptor activity during prostate cancer progression

Mounting evidence suggests that the androgen receptor (AR) plays a critical role in prostate cancer (PCa) progression. However, the molecular mechanisms by which the AR regulates cell proliferation in androgen-dependent and castration-resistant PCa are incompletely understood. We report here that calcium/calmodulin-dependent kinase kinase 2 (CaMKK2) expression increases during progression of human PCa and becomes nuclear or perinuclear in advanced disease. In a mouse model of PCa (transgenic adenocarcinoma of mouse prostate, TRAMP), CaMKK2 expression increases with progression and is predominantly nuclear. CaMKK2 expression is markedly higher in the AR-expressing, tumorigenic cell line LNCaP compared to cell lines which are AR-nonexpressing and/or nontumorigenic. Silencing of CaMKK2 expression by RNA interference (RNAi) reduces cell proliferation in LNCaP cells but not in AR-non-expressing PC3 cells. In LNCaP cells, dihydrotestosterone (DHT) induces CaMKK2 mRNA and protein expression while androgen withdrawal suppresses CaMKK2 expression. RNAi of CaMKK2 reduces expression of the AR target, prostate specific antigen (PSA) and the cell cycle regulators cyclin D1 and phospho-pRB. Our results suggest that in PCa progression, CaMKK2 is transcriptionally induced by the AR to maintain AR activity, AR-dependent cell cycle control and continued cell proliferation. Citation Format: Loukia G. Karacosta, Barbara A. Foster, Azabdaftari Gissou, David M. Feliciano, Arthur M. Edelman. CaMKK2 regulates cellular proliferation and androgen receptor activity during prostate cancer progression [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 A10.