Kelly A. Conrads

An integrated understanding of the physiological response to elevated extracellular phosphate

Recent studies have suggested that changes in serum phosphate levels influence pathological states associated with aging such as cancer, bone metabolism, and cardiovascular function, even in individuals with normal renal function. The causes are only beginning to be elucidated but are likely a combination of endocrine, paracrine, autocrine, and cell autonomous effects. We have used an integrated quantitative biology approach, combining transcriptomics and proteomics to define a multi‐phase, extracellular phosphate‐induced, signaling network in pre‐osteoblasts as well as primary human and mouse mesenchymal stromal cells. We identified a rapid mitogenic response stimulated by elevated phosphate that results in the induction of immediate early genes including c‐fos. The mechanism of activation requires FGF receptor signaling followed by stimulation of N‐Ras and activation of AP‐1 and serum response elements. A distinct long‐term response also requires FGF receptor signaling and results in N‐Ras activation and expression of genes and secretion of proteins involved in matrix regulation, calcification, and angiogenesis. The late response is synergistically enhanced by addition of FGF23 peptide. The intermediate phase results in increased oxidative phosphorylation and ATP production and is necessary for the late response providing a functional link between the phases. Collectively, the results define elevated phosphate, as a mitogen and define specific mechanisms by which phosphate stimulates proliferation and matrix regulation. Our approach provides a comprehensive understanding of the cellular response to elevated extracellular phosphate, functionally connecting temporally coordinated signaling, transcriptional, and metabolic events with changes in long‐term cell behavior. J. Cell. Physiol. 228: 1536–1550, 2013.

Elevated AKAP12 in paclitaxel-resistant serous ovarian cancer cells is prognostic and predictive of poor survival in patients.

A majority of high-grade (HG) serous ovarian cancer (SOC) patients develop resistant disease despite high initial response rates to platinum/paclitaxel-based chemotherapy. We identified shed/secreted proteins in preclinical models of paclitaxel-resistant human HGSOC models and correlated these candidate proteins with patient outcomes using public data from HGSOC patients. Proteomic analyses of a HGSOC cell line secretome was compared to those from a syngeneic paclitaxel-resistant variant and from a line established from an intrinsically chemorefractory HGSOC patient. Associations between the identified candidate proteins and patient outcome were assessed in a discovery cohort of 545 patients and two validation cohorts totaling 795 independent SOC patients. Among the 81 differentially abundant proteins identified (q < 0.05) from paclitaxel-sensitive vs -resistant HGSOC cell secretomes, AKAP12 was verified to be elevated in all models of paclitaxel-resistant HGSOC. Furthermore, elevated AKAP12 transcript expression was associated with worse progression-free and overall survival. Associations with outcome were observed in three independent cohorts and remained significant after adjusted multivariate modeling. We further provide evidence to support that differential gene methylation status is associated with elevated expression of AKAP12 in taxol-resistant ovarian cancer cells and ovarian cancer patient subsets. Elevated expression and shedding/secretion of AKAP12 is characteristic of paclitaxel-resistant HGSOC cells, and elevated AKAP12 transcript expression is a poor prognostic and predictive marker for progression-free and overall survival in SOC patients.

NUAK1 (ARK5) Is Associated with Poor Prognosis in Ovarian Cancer

Background and objective Nua kinase 1 (NUAK1) was identified in multigene signatures of survival and suboptimal debulking in high-grade serous ovarian cancer (HGSOC). This study investigates the individual clinical and biologic contributions of NUAK1 in HGSOC patients and cell lines. Methods Public transcript expression, clinical, and outcome data were used to interrogate the relationship between NUAK1 and clinicopathologic factors and patient outcomes including progression-free survival (PFS) and molecular subtypes using logistic and Cox modeling. Analysis of NUAK1 transcript expression was performed in primary tumors from 34 HGSOC patients with < or ≥2 years PFS. The impact of silencing NUAK1 by RNA interference (RNAi) on the migratory potential and chemosensitivity of SOC cells was assessed in vitro. Results Elevated NUAK1 transcript expression was associated with worse PFS (hazard ratio = 1.134), advanced stage (odds ratio, OR = 1.7), any residual disease (OR = 1.58), and mesenchymal disease subtype (OR = 7.79 ± 5.89). Elevated NUAK1 transcript expression was observed in HGSOC patients with < vs. ≥2 years PFS (p < 0.045). RNAi-mediated silencing of NUAK1 expression attenuated migration of OV90 and E3 HGSOC cells in vitro, but did not modulate sensitivity to cisplatin or paclitaxel. Conclusion Elevated NUAK1 was associated with poor survival as well as advanced stage, residual disease after cytoreductive surgery and mesenchymal molecular subtype. NUAK1 impacted migration, but not chemosensitivity, in vitro. Additional studies are needed to further develop the concept of NUAK1 as a clinically deployable biomarker and therapeutic target in HGSOC.

Identification and functional characterization of a novel bipartite nuclear localization sequence in ARID1A.

AT-rich interactive domain-containing protein 1A (ARID1A) is a recently identified nuclear tumor suppressor frequently altered in solid tumor malignancies. We have identified a bipartite-like nuclear localization sequence (NLS) that contributes to nuclear import of ARID1A not previously described. We functionally confirm activity using GFP constructs fused with wild-type or mutant NLS sequences. We further show that cyto-nuclear localized, bipartite NLS mutant ARID1A exhibits greater stability than nuclear-localized, wild-type ARID1A. Identification of this undescribed functional NLS within ARID1A contributes vital insights to rationalize the impact of ARID1A missense mutations observed in patient tumors.

Abstract 214: ARID1A and ARID1B dependent proteomics

Protein sub-units of the SWI/SNF nucleosome and chromatin remodeling complexes are frequently mutated in cancer. The ARID1A and ARID1B DNA interacting component are among these mutated proteins. Alterations in ARID1A have been reported in breast, colon, lung, kidney, pancreatic, bladder, cervical, ovarian and uterine cancers whereas ARID1B mutation is less common. Additional data suggests ARID1A mutant cancers are dependent on functional ARID1B and that targeting ARID1B may be a therapeutic strategy. However, ARID1A and 1B are often co-inactivated in aggressive un/dedifferentiated carcinomas of the ovary and endometrium suggesting tumor suppressive functions might exist not only in ARID1A but also in 1B. In this study, we examined the effects of restoring ARID1A or 1B in an undifferentiated endometrial adenocarcinoma cell line harboring inactivating mutations in both genes. ACI-98 cells derived from a stage IV undifferentiated endometrial cancer were found to lack expression of ARID1A and 1B protein. We subsequently identified two individual truncating mutations in ARID1A and 1B by genomic DNA and cDNA sequencing indicating no wild-type message was expressed. Restoration of ARID1A in ACI-98 cells using a Tet-on system revealed remarkable growth inhibition, however, ARID1B restoration showed only a moderate cell growth inhibition with cells taking on a flattened phenotype. Clones of ARID1A/1B restored cells were treated w/wo doxycycline (Dox) for 48h a time point prior to cell death or morphologic changes and protein changes catalogued using LC MS/MS. We identified 771 and 1168 differentially expressed proteins (z-score Citation Format: Yutaka Shoji, Kumiko Kato-Shoji, Kelly A. Conrads, Rusheeswar Challa, Brian L. Hood, Nicholas W. Bateman, Thomas P. Conrads, John I. Risinger. ARID1A and ARID1B dependent proteomics [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 214. doi:10.1158/1538-7445.AM2017-214

Abstract 2359: Inhibition of the ATR kinase enhances therapeutic efficacy of cisplatin in ATM low uterine carcinosarcoma cells

Objective: Uterine carcinosarcoma (UCS) is an aggressive malignancy, making up less than 5% of uterine cancers. Pharmacologic inhibition of ATR kinase synergistically enhances the ability of cisplatin to kill carcinoma cells of many solid tumor types and is further enhanced in ATM low carcinoma cells. It is unknown whether ATR inhibition (ATRi) offers a therapeutic opportunity in UCS. The goal of this study was to evaluate ATM expression in UCS tumors and determine the response of an ATM low UCS model to ATRi + cisplatin combination. Methods: ATM expression was evaluated in full tissue sections from 74 UCS tumors by immunohistochemistry (IHC) with an ATM specific monoclonal antibody (clone Y170, Abcam), and was categorized as negative vs any positive staining in the carcinomatous (C) vs sarcomatous (S) components. In vitro models of UCS: SK-UT-1, SK-UT-1B, KLE, and RL95-2 cell lines were treated with an ATRi (AZD6738), cisplatin, and ATRi + cisplatin. ATM low models were generated in RL95-2 cells using short hairpin RNA (shRNA) lentivirus targeting ATM and a non-target control. Cells were subjected to cisplatin and ATRi dose response analyses. Results: Negative IHC expression of ATM protein was observed in 24% of the C vs 31% of the S components of the 74 UCS cases (Table 1). ATRi increased the sensitivity of uterine carcinosarcoma cell line models to cisplatin. Western blot confirmed a 57% knockdown in ATM in RL95-2 cells infected with shRNA for ATM and treatment with ATRi +cisplatin therapy showed ~20% increase in cell death in ATM low vs control cells lines, p=0.02. Conclusions: IHC analyses of UCS tumors showed an average of ~27% of tumors have loss of ATM in C and S components. Preliminary evidence shows that ATRi increases the sensitivity of UCS cell models to cisplatin therapy, which is further increased in ATM low uterine carcinoma cells. These findings suggest a novel therapeutic opportunity for ATRi + cisplatin therapy in UCS patients with low ATM expressing tumors. Citation Format: Emily R. Penick, Paulette Mhawech-Fauceglia, Nicholas Bateman, Kelly Conrads, Tracy Litzi, Chunqiao Tian, Chad A. Hamilton, Kathleen Darcy, George Maxwell, Thomas Conrads. Inhibition of the ATR kinase enhances therapeutic efficacy of cisplatin in ATM low uterine carcinosarcoma cells [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 2359. doi:10.1158/1538-7445.AM2017-2359

Abstract A19: Cytoplasmic ARID1A and poor outcome in ovarian cancer patients.

Objective: Frequent loss of function mutations in the chromatin remodeling protein and tumor suppressor, AT-rich interactive domain-containing protein 1A (ARID1A), have been noted in ovarian cancers, particularly clear-cell and mucinous histotypes. We initially investigated the relationship between differential ARID1A protein levels and patient survival in a well-annotated ovarian cancer tissue microarray. Our analyses revealed distinct cellular localization patterns for ARID1A using moderately stringent assay conditions and we observed a relationship between cytoplasmic staining of ARID1A and poor disease outcome. We further investigated the relevance of cytoplasmic ARID1A in vitro in a cell line model of ARID1A-mutated, clear-cell ovarian cancer and found that ectopic expression of a cytoplasm-localized variant of ARID1A enhances cellular proliferation relative to wild type ARID1A restored cells. Methods: Tissue microarrays representing a natural distribution of ovarian cancer histotypes (N=259) were stained for ARID1A and scored. Four patient subgroups were further defined based on the absence or presence of distinct ARID1A staining in nuclear and/or cytoplasmic tumor cell compartments. Relationships between ARID1A staining pattern and patient outcome were investigated using univariate and multivariate Cox regression modeling with Wald testing and Kaplan-Meier method with log-rank testing. Nuclear localization sequence variants of ARID1A were generated and phenotypic assessments of cellular proliferation were performed following ectopic expression of cytoplasm-localized versus wild type, i.e. nuclear-localized, ARID1A in a cell line model of ARID1A-mutated, clear cell ovarian cancer cells (TOV21G). Results: Using a moderately stringent IHC assay for ARID1A, loss of nuclear ARID1A was observed in 7% of ovarian cancer patients, and women with versus without loss of nuclear ARID1A had similar survival. Prevalent cytoplasmic staining was observed in 14% of cases overall and was more common in mucinous (50%), clear cell (42%) and serous (11%) adenocarcinomas than other histotypes (p=0.002). Prevalent cytoplasmic ARID1A was not only associated with an increased risk of death in the entire cohort (HR=1.67, 95% CI=1.137-2.454, p=0.009), but was also an independent predictor of worse survival (adjusted HR=1.530, 95% CI=1.017-2.301, p=0.041) after adjusting for patient age at diagnosis, site of disease, stage, cell type and grade. Comparative in vitro analyses of cytoplasmic and wild type ARID1A variants, ectopically-expressed in clear-cell ovarian cancer cells revealed that cytoplasmic ARID1A confers an increased proliferative phenotype relative to wild type ARID1A restored cells. Conclusions: ARID1A cellular localization and abundance varied across ovarian cancer histotypes and cytoplasmic localization was indicative of worse outcome. Our in vitro studies suggest cytoplasmic ARID1A increases the proliferative potential of clear-cell ovarian tumor cells and provides phenotypic evidence supporting the poor disease outcomes we observe in patients exhibiting cytoplasmic ARID1A. These findings support further investigations combining assessments of ARID1A cellular localization and abundance patterns towards the identification of ovarian cancer patients with an elevated risk to experience poor disease outcome. Citation Format: Nicholas W. Bateman, Kevin A. Byrd, Kelly A. Conrads, John I. Risinger, Carl Morrison, Kunle Odunsi, Chad A. Hamilton, G. Larry Maxwell, Kathleen M. Darcy, Thomas P. Conrads. Cytoplasmic ARID1A and poor outcome in ovarian cancer patients. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A19.

Abstract 2747: Pharmacological inhibition of the DNA damage response kinases, ATR (Ataxia telangiectasia and Rad3 related) and ATM (Ataxia telangiectasia mutated), broadly sensitizes diverse subtypes of gynecological cancer cells to ionizing radiation

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Objectives: The management of gynecological malignancies includes exposure of tumor cells to genotoxic insults, such as ionizing radiation (IR), and focuses on damaging cellular DNA and inducing subsequent cell death. As cells possess innate mechanisms to repair DNA damage, this study focused on combining IR with pharmacological inhibition of key mediators of DNA repair, ATR (Ataxia telangiectasia and Rad3 related) and ATM (Ataxia telangiectasia mutated), to further sensitize gynecological cancer cells to this mode of genotoxic stress. Methods: Clonogenic survival assays: A panel of human cell line models of ovarian, endometrial and cervical cancer were treated with drug vehicle, 5.0 µM ATR inhibitor (ETP-46464), 10.0 µM ATM inhibitor (KU55933) or a combination of ATRi and ATMi, prior to exposure to IR doses up to 6.0 Gy. Drug was removed four hours later and resulting colonies were counted when mean colony size was ≥ 50 cells. Western blot: ATM, ATR and downstream canonical signaling effectors were assessed in cell lysates harvested from representative cell line models of ovarian (A2780), endometrial (HEC1B) and cervical (HELA) cancers, one hour following treatment with inhibitors and IR exposure (2.0 Gy only) as described above. Results: Clonogenic survival assays revealed that inhibition of ATR and ATM increased sensitization to IR across all cell line models of gynecological cancer assessed. This effect was further increased with combined ATR and ATM inhibitor treatments. Western blot analyses revealed activation of ATM and ATR signaling in response to IR via increases in phospho-ATM (Ser1981), p-Chk1 (Ser345) and p-Chk2 (T68) levels. Activation of Chk1, a downstream effector of ATR, was attenuated in ATRi-treated conditions and activation of Chk2, a downstream effector of ATM, were reduced in ATMi-treated conditions. Conclusions: These studies revealed that inhibition of the DNA damage response kinases, ATR and ATM, markedly sensitizes diverse gynecological cancer subtypes to IR. They provide evidence to support further consideration of therapies pairing modulation of DNA damage signaling with IR in the treatment of gynecological malignancies. Citation Format: Nicholas Bateman, Pang-ing Teng, Kelly Conrads, Chad Hamilton, George Maxwell, Christopher Bakkenist, Thomas Conrads. Pharmacological inhibition of the DNA damage response kinases, ATR (Ataxia telangiectasia and Rad3 related) and ATM (Ataxia telangiectasia mutated), broadly sensitizes diverse subtypes of gynecological cancer cells to ionizing radiation. [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 2747. doi:10.1158/1538-7445.AM2014-2747

Abstract 1570: ARID1A regulation of ATAD2 in gynecologic cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA ARID1A the DNA binding component of the SWI/SNF nucleosome and chromatin remodeling complex has recently been identified as a tumor suppressor and inactivating mutations have been reported in several tumor types including uterine and ovarian cancer. Approximately 30% of ovarian endometrioid cancers (OEC), 50% of clear cell ovarian cancers (OCCC) and 40% of uterine endometrial cancers harbor ARID1A inactivating mutations. Little is known regarding the subunit composition of SWI/SNF complexes that contain ARID1A in ovarian and uterine cells and less is known regarding the transcriptional and functional consequences of ARID1A loss. In order to further explore the function of ARID1A in gynecologic cancers we restored ARID1A in ACI-98 (undifferentiated uterine cancer) an ARID1A negative and TP53 positive cell line using an inducible Tet-on system. Restoration of ARID1A induced apoptosis in ACI-98 cells after 48h doxycycline (Dox) treatment. We examined the proteome of these cells and catalogued the protein changes following ARID1A complementation using LC MS/MS. Specifically we identified 523 highly significantly differentially expressed proteins (z-score <0.01) from this analysis. Among these was the AAA domain containing 2 (ATAD2) that was dramatically down-regulated in ARID1A restored cells. ATAD2 is a highly conserved protein normally expressed in germ cells but is also over-expressed in some cancers. Affymetrix gene expression results also showed that ATAD2 is over-expressed in some ovarian and uterine cancers compared to normal controls. We further examined the clinical consequence of ATAD2 expression in ovarian cancers using the Kaplan-Meier plots website (http://kmplot.com) and found that the high-expression group has a worse overall survival than those in the low-expression group. ATAD2 associates via its bromodomain with histone H3 and it is known to act as a co-factor for E2Fs, MYC, androgen and estrogen receptors and whose over-expression drives the expression of target genes that induce cell proliferation and resistance to apoptosis. qRT-PCR results indicated that transcription of ATAD2 was not changed by restoration of ARID1A, moreover, anti-ARID1A ChIP-sequence revealed that SWI/SNF-ARID1A is not binding to the promoter of ATAD2. From these results, we suspect that the down regulation of ATAD2 is through a non-transcriptional mechanism. In addition, ARID1A-IP-MS result suggested that ARID1A is itself making complexes with ATAD2. We performed ATAD2 and ARID1A immunohistochemistry on a set of OCCC and OEC primary cancers and found that all cases with ARID1A negative staining preferentially over-express ATAD2. In summary our data indicate that ARID1A decreases levels of ATAD2, that ARID1A-negative OCCC and OEC are a subset of ovary cancer that preferentially overexpress ATAD2, and that ATAD2 over-expressing cancers have worse survival than weak expressers. Citation Format: Yutaka Shoji, Kelly A. Conrads, Rusheeswar Challa, Brian L. Hood, Guisong Wang, Kathleen M. Darcy, Chad A. Hamilton, George Larry Maxwell, Thomas P. Conrads, John I. Risinger. ARID1A regulation of ATAD2 in gynecologic 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 1570. doi:10.1158/1538-7445.AM2014-1570

Abstract 990: Metastasis suppressor CD82/KAI1 expression is dependent on functional SWI/SNF ARID1A/B

CD82/KAI1 was one of the first metastasis suppressors identified and is capable of inhibiting the metastatic process in orthotopic tumor models. The expression of the CD82/KAI1 is down-regulated during tumor progression in many tumor types including prostate, breast, lung, bladder, and pancreatic cancers, and this down regulation appears to be at the level of transcription or post-transcription. The gene is not mutated, deleted or silenced by promoter methylation. CD82/KAI1 is also down regulated in advanced gynecologic cancers where CD82/KAI1 loss is associated with the poor overall survival in ovarian cancer. The mechanisms controlling the level of CD82/KAI1 expression remains unclear particularly the widespread loss in advanced cancers. The p53 tumor suppressor (TP53) can directly activate the CD82/KAI1 gene by interacting with the 5-prime upstream region of the CD82/KAI1 gene. Mutant TP53 is unable to bind this promoter site and fails to support CD82/KAI1 transcription. Taken together these data support a direct relationship between TP53 and CD82/KAI1 genes and suggested that the loss of TP53 function, which is common in many types of advanced cancer can lead to downregulation of CD82/KAI1, which then may result in metastases. However many cancers without TP53 mutation also exhibit loss of CD82/KAI1 suggesting that the mechanism for CD82/KAI1 loss is not completely explained by mutant TP53. Furthermore the mutant TP53 state and CD82/KAI1 loss is not strongly correlated in ovarian cancer. In some sarcomas the mechanism of CD82/KAI1 loss is related to the increase of the gp-78 E3 ligase and increased proteasomal degradation. Therefore the regulation of CD82/KAI1 in cells is likely complex. The AT Rich DNA binding protein ARID1A gene is frequently mutated in endometrial and ovarian cancers. In order to explore the functions of ARID1A in these cancers we complemented mutant ARID1A in the ACI-98 endometrial cancer cell line. We utilized a doxycycline inducible system and catalogued the protein changes following ARID1A complementation using LC MS/MS. Among the most differentially expressed proteins was CD82/KAI1. Levels of CD82 were dramatically increased following ARID1A induction. ACI-98 cells were found to be TP53 wild-type and CD82 expression null. QRT-PCR assays confirmed the dramatic increase in CD82/KAI1 mRNA levels and western blots also confirmed restoration of CD82/KAI1 protein following Doxycycline induced ARID1A restoration. We performed ChIP and confirmed that ARID1A was bound to the CD82/KAI1 promoter region close to the TP53 interaction area. Interestingly ACI-98 cells are mutant in both ARID1 homologs A and B. Ectopic expression of ARID1B was also able to restore expression of CD82/KAI1. These data suggest that the SWI/SNFARID1A/B complex is necessary for chromatin remodeling and expression of the CD82/KAI1 gene. Citation Format: Rusheeswar Challa, Yutaka Shoji, Kelly A. Conrads, Brian L. Hood, Guisong Wang, Kathleen M. Darcy, Chad A. Hamilton, George Larry Maxwell, Thomas P. Conrads, John I. Risinger. Metastasis suppressor CD82/KAI1 expression is dependent on functional SWI/SNF ARID1A/B. [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 990. doi:10.1158/1538-7445.AM2014-990