Nicholas W. Bateman

Intestinal tumor progression is associated with altered function of KLF5

Krüppel-like transcription factors have been linked to cell growth regulation and tumorigenesis in a number of systems. In the intestinal epithelium, expression of KLF5 (IKLF/BTEB2) is limited to proliferating crypt cells, indicating a growth-promoting role. Consistent with this role, we demonstrate that expression of KLF5 in non-transformed intestinal epithelial cells (ileal IEC-18 and Immorto-Min Colon Epithelial (IMCE) cells) enhances colony formation, cyclin D1 transcription, and cell growth. However, in contrast to these effects in non-transformed cells, KLF5 reduced colony number, failed to enhance cyclin D1 transcription, and was negatively correlated with cell growth in colon cancer cell lines. The relationship between tumor progression and KLF5 was further investigated using Ras-mediated transformation of IEC-18 and IMCE cells as syngeneic models. Ras-transformation recapitulated differences in the effects of KLF5 on cell growth and cyclin D1 transcription, providing a direct link between intestinal tumor progression and altered function of KLF5. Ras-transformation also markedly down-regulated KLF5; further analysis indicated that reduced expression of KLF5 mRNA and destabilization of KLF5 protein occur in intestinal tumors. Reduced levels of KLF5 mRNA were also detected in APCmin mouse and human familial adenomatous polyposis adenomas compared with normal crypt epithelium, indicating that down-regulation of KLF5 is an early event in intestinal tumorigenesis in vivo. Collectively, these data indicate that intestinal tumor progression is associated with a change in the growth-related functions of KLF5 and that intestinal tumors down-regulate KLF5 expression by multiple mechanisms.

Advances in proximal fluid proteomics for disease biomarker discovery.

Although serum/plasma has been the preferred source for identification of disease biomarkers, these efforts have been met with little success, in large part due the relatively small number of highly abundant proteins that render the reliable detection of low abundant disease-related proteins challenging due to the expansive dynamic range of concentration of proteins in this sample. Proximal fluid, the fluid derived from the extracellular milieu of tissues, contains a large repertoire of shed and secreted proteins that are likely to be present at higher concentrations relative to plasma/serum. It is hypothesized that many, if not all, proximal fluid proteins exchange with peripheral circulation, which has provided significant motivation for utilizing proximal fluids as a primary sample source for protein biomarker discovery. The present review highlights recent advances in proximal fluid proteomics, including the various protocols utilized to harvest proximal fluids along with detailing the results from mass spectrometry- and antibody-based analyses.

Differential proteomic analysis of late-stage and recurrent breast cancer from formalin-fixed paraffin-embedded tissues.

The heterogeneity of breast cancer requires the discovery of more incisive molecular tools that better define disease progression and prognosis. Proteomic analysis of homogeneous tumor cell populations derived by laser microdissection from formalin-fixed, paraffin-embedded (FFPE) tissues has proven to be a robust strategy for conducting retrospective cancer biomarker investigations. We describe an MS-based analysis of laser microdissected cancerous epithelial cells derived from twenty-five breast cancer patients at defined clinical disease stages with the goal of identifying protein abundance characteristics indicative of disease progression and recurrence. Comparative analysis of stage 0 and stage III patients revealed 113 proteins that significantly differentiated these groups and included known factors associated with disease pathogenesis, such as CDH1 and CTNNB1, as well as those previously implicated in breast cancer, such as TSP-1. Similar analyses of patients presenting with stage II disease that did or did not exhibit recurrence two years postdiagnosis revealed 42 proteins that significantly differentiated these subgroups and included IRS-1 and PARK7. These data provide evidence supporting the utility of FFPE tissues for functional proteomic analyses and protein biomarker discovery and yielded protein candidates indicative of disease stage and recurrence in breast cancer that warrant further investigation for diagnostic utility and biological relevance.

PKCα tumor suppression in the intestine is associated with transcriptional and translational inhibition of cyclin D1

Alterations in PKC isozyme expression and aberrant induction of cyclin D1 are early events in intestinal tumorigenesis. Previous studies have identified cyclin D1 as a major target in the antiproliferative effects of PKCalpha in non-transformed intestinal cells; however, a link between PKC signaling and cyclin D1 in colon cancer remained to be established. The current study further characterized PKC isozyme expression in intestinal neoplasms and explored the consequences of restoring PKCalpha or PKCdelta in a panel of colon carcinoma cell lines. Consistent with patterns of PKC expression in primary tumors, PKCalpha and delta levels were generally reduced in colon carcinoma cell lines, PKCbetaII was elevated and PKCepsilon showed variable expression, thus establishing the suitability of these models for analysis of PKC signaling. While colon cancer cells were insensitive to the effects of PKC agonists on cyclin D1 levels, restoration of PKCalpha downregulated cyclin D1 by two independent mechanisms. PKCalpha expression consistently (a) reduced steady-state levels of cyclin D1 by a novel transcriptional mechanism not previously seen in non-transformed cells, and (b) re-established the ability of PKC agonists to activate the translational repressor 4E-BP1 and inhibit cyclin D1 translation. In contrast, PKCdelta had modest and variable effects on cyclin D1 steady-state levels and failed to restore responsiveness to PKC agonists. Notably, PKCalpha expression blocked anchorage-independent growth in colon cancer cells via a mechanism partially dependent on cyclin D1 deficiency, while PKCdelta had only minor effects. Loss of PKCalpha and effects of its re-expression were independent of the status of the APC/beta-catenin signaling pathway or known genetic alterations, indicating that they are a general characteristic of colon tumors. Thus, PKCalpha is a potent negative regulator of cyclin D1 expression and anchorage-independent cell growth in colon tumor cells, findings that offer important perspectives on the frequent loss of this isozyme during intestinal carcinogenesis.

Quantitative Mass Spectrometry by Isotope Dilution and Multiple Reaction Monitoring (MRM)

Selected reaction monitoring (SRM) is used in molecular profiling to detect and quantify specific known proteins in complex mixtures. Using isotope dilution (Barnidge et al., Anal Chem 75(3):445-451, 2003) methodologies, peptides can be quantified without the need for an antibody-based method. Selected reaction monitoring assays employ electrospray ionization mass spectrometry (ESI-MS) followed by two stages of mass selection: a first stage where the mass of the peptide ion is selected and, after fragmentation by collision-induced dissociation (CID), a second stage (tandem MS) where either a single (e.g., SRM) or multiple (multiple reaction monitoring, MRM) specific peptide fragment ions are transmitted for detection. The MRM experiment is accomplished by specifying the parent masses of the selected endogenous and isotope-labeled peptides for MS/MS fragmentation and then monitoring fragment ions of interest, using their intensities/abundances and relative ratios to quantify the parent protein of interest. In this example protocol, we will utilize isotope dilution MRM-MS to quantify in absolute terms the total levels of the protein of interest, ataxia telangiectasia mutated (ATM) serine/threonine protein kinase. Ataxia telangiectasia mutated (ATM) phosphorylates several key proteins that initiate activation of the DNA damage checkpoint leading to cell cycle arrest.

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 5277: Proteome and transcriptome alterations in black endometrial cancer patients correlate with poor disease outcome

Objective: Black endometrial cancer patients are more than twice as likely to die from their disease as White patients. This study sought to identify alterations in the proteome and transcriptome of primary tumor tissues from White and Black endometrioid endometrial cancer (EEC) patients associated with differential outcome. Methods: An integrated proteomic and transcriptomic analysis (LC-MS/MS and RNA-seq) was performed on White (n=13) and Black (n=17) EEC patient tissues. Significant and concordantly altered protein and transcript candidates were validated against publicly available RNA-seq data (TCGA UCEC) from White (n=216) and Black (n=49) EEC patients. Validated candidates were further correlated with overall (OS, n=356 White and Black patients) and progression-free survival (PFS, n=331 White and Black patients) to identify candidates significantly associated with differential disease outcome. Alterations of outcome-associated candidates were validated in an independent cohort of White (n=115) and Black (n=17) EEC patient transcript expression data. Results: We identified and validated 89 proteins and transcripts significantly altered between White vs Black EEC patients. Pathway analyses revealed candidates elevated in White EEC patients correlated with marked activation of molecular signaling pathways regulating viral infection, but inhibition of those regulating cell death and necrosis. Candidates elevated in Black EEC patients largely correlated with activation of cell viability and nucleic acid metabolism, but inhibition of cell death, glucose metabolism disorder and inflammatory signaling. Correlation with patient outcome measures revealed 11 candidates significantly associated with differential OS and 8 candidates with differential PFS in EEC patients. All outcome-associated candidates elevated in White patients significantly correlated with a low risk of poor OS and poor PFS (Hazard Ratio (HR) 1, Wald p-value Conclusions: Our analyses identified and confirmed molecular alterations between White and Black EEC patients, including outcome-associated candidates largely supportive of better outcome in White patients, but poor outcome in Black patients. These findings define molecular alterations in White and Black EEC patients consistent with the historic disparity of poor outcome for Black patients warranting further investigation of these candidates in Black EEC disease pathology. Citation Format: Nicholas W. Bateman, Elizabeth Dubil, Guisong Wang, Brian L. Hood, Tracy Litzi, Julie Oliver, Kathleen M. Darcy, Chad A. Hamilton, Thomas P. Conrads, George L. Maxwell. Proteome and transcriptome alterations in black endometrial cancer patients correlate with poor disease outcome [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 5277. doi:10.1158/1538-7445.AM2017-5277

Abstract 3758: Inhibition of ATR, but not ATM, sensitizes gynecologic cancer cells to cisplatin

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Resistance to platinum-based therapies remains a major hurdle in the management of gynecologic (GYN) cancer especially in ovarian cancer. Platinum such as cisplatin damages DNA by inducing DNA crosslinks that stalls DNA replication forks where significant accumulation of single-stranded DNA from persistently stalled replication forks could ultimately lead to double strand breaks and activation of apoptosis. Ataxia telangiectasia mutated (ATM) and ATM and Rad-3-related (ATR) are two main DNA damage response (DDR) protein kinases that recognize genotoxic stress and function to initiate cell cycle arrest and DNA repair mechanisms. We hypothesized by combining genotoxic stress with inhibition of DDR kinases ATR and/or ATM, therapeutic response of GYN cancer cells to platinum-based chemotherapy could be improved. To test our hypothesis, we assessed cell survival of multiple GYN cell lines including ovarian (A2780, A2780-CP20, OVCAR3), endometrial (KLE, HEC1B), and cervical (HELA, SIHA) carcinoma cells exposed to cisplatin along with ATR inhibitor (ETP-46464) and/or ATM inhibitor (KU55933). We observed inhibition of ATR significantly enhanced cisplatin induced cell death in all seven cell lines tested, resulting in 65 - 96% increased sensitivity to cisplatin. Inhibition of ATM did not sensitize GYN cancer cells to cisplatin and cells were not further sensitized by co-inhibition of ATM and ATR beyond that observed by inhibition of ATR alone. DDR signaling was assessed by immunoblotting in cells exposed to cisplatin with or without the presence of ETP-46464 and/or KU55933 where elevated levels of phospho-ATM (Ser1981), phospho-Chk2 (Thr68) and phospho-Chk1 (Ser345) observed in cisplatin treated cells were attenuated in cells that were co-treated with cisplatin and ETP-46464. In addition, increased levels of cleaved PARP1 and caspase 3 were observed in cisplatin-treated, ATR-inhibited cells, suggesting the enhancement of cisplatin induced cell death with ATR inhibition occurs through elevated apoptosis. Further, no differential effect was observed in GYN cancer cells harboring wild type (A2780, OVCAR3, HELA, SIHA) or mutant (CP20, KLE, HEC1B) TP53. These data support further investigation of pharmacologic inhibitors of ATR in combination with existing platinum based therapeutics for treating GYN cancer. Citation Format: Pang-Ning Teng, Nicholas W. Bateman, Chad A. Hamilton, G. Larry Maxwell, Christopher J. Bakkenist, Thomas P. Conrads. Inhibition of ATR, but not ATM, sensitizes gynecologic cancer cells to cisplatin. [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 3758. doi:10.1158/1538-7445.AM2014-3758

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