Elizabeth M. Matthew

CARPs Are Ubiquitin Ligases That Promote MDM2-independent p53 and Phospho-p53ser20 Degradation

Caspase 8/10-associated RING proteins (CARPs) are a recently described family of protein ubiquitin ligases that interact with and negatively regulate death receptor-mediated apoptosis. Because CARPs are overexpressed in cancer and their silencing reduces cell viability and sensitizes tumor cells to chemotherapeutic agents, we investigated their relationship to p53 tumor suppressor signaling. p53 is a major determinant of chemosensitivity, and its levels are increased following DNA damage through N-terminal phosphorylation and inhibition of degradation. Although p53 is well known to be negatively regulated by several ubiquitin ligases including MDM2, none are known to target phosphorylated p53 for degradation. CARPs physically interact with and ubiquitinate p53, targeting it for degradation in the absence of MDM2. Serine 20-phosphorylated p53 is also ubiquitinated by CARPs. CARP silencing stimulates p53 expression and promotes downstream effects, including transcriptional activation and tumor suppression.

The p53 target Plk2 interacts with TSC proteins impacting mTOR signaling, tumor growth and chemosensitivity under hypoxic conditions

Tuberous sclerosis complex 1 (TSC1) inhibits mammalian target of rapamycin (mTOR), a central promotor of cell growth and proliferation. The protein product of the TSC1 gene, hamartin (referred to as TSC1) is known to interact with Polo-like kinase 1 (Plk1) in a cell cycle regulated, phosphorylation-dependent manner. We hypothesized that the p53 target gene, Plk2, is a tumor suppressor, mediating its tumor suppressor function through interactions with TSC1 that facilitate TSC1/2 restraint of mTOR under hypoxic stress. We found that human lung tumor cells deficient in Plk2 grew larger than control tumors, and that Plk2 interacts with endogenous TSC1 protein. Additionally, C-terminal Plk2-GST fusion protein bound both TSC1 and TSC2 proteins. TSC1 levels were elevated in response to Adriamycin and cells transiently over-expressing Plk2 demonstrated decreased phosphorylation of the downstream target of mTOR, ribosomal protein p70S6 kinase during hypoxia. Plk2 levels were inversely correlated with cytoplasmic p70S6K phosphorylation. Plk2 levels did not increase in response to DNA damage (Adriamycin, CPT-11) when HCT 116 and H460 cells were exposed to hypoxia. TSC1-deficient mouse embryonic fibroblasts with TSC1 added back demonstrated decreased S6K phosphorylation, which was further decreased when Plk2 was transiently over-expressed. Interestingly, under normoxia, Plk2 deficient tumor cells demonstrated increased apoptosis in response to various chemotherapeutic agents including CPT-11 but increased resistance to apoptotic death after CPT-11 treatment under hypoxia, and tumor xenografts comprised of these Plk2-deficient cells were resistant to CPT-11. Our results point to a novel Plk2-TSC1 interaction with effects on mTOR signaling during hypoxia, and tumor growth that may enable targeting Plk2 signaling in cancer therapy.

Replication stress, defective S-phase checkpoint and increased death in Plk2-deficient human cancer cells.

We previously reported that the Polo-like Kinase 2 gene (Plk2/Snk) is a direct target for transcriptional regulation by p53 and that silencing Plk2 sensitizes cancer cells to Taxol-induced apoptosis. Our goals have been to better understand why Plk2 is regulated by p53 and how Plk2 signals protection from cell death through checkpoint activation. We found that following knock-down of Plk2 in wild-type p53 expressing H460 human non-small cell lung cancer cells there was a significant increase in cell death observed in aphidicolin-treated cells and a further increase after release from aphidicolin-block. The highest levels of cell death were observed when Plk2-deficient cells were released from both aphidicolin and etoposide treatment. These results suggested that a defective S-phase checkpoint may contribute to enhanced sensitivity of Plk2-deficient cells to replication stress. Consistent with this hypothesis, we observed higher levels of Serine 139 H2AX phosphorylation in Plk2-deficient as compared to control cells before and after aphidicolin treatment indicating that there is more DNA damage when Plk2 is depleted. We also observed higher levels of Chk1 protein in Plk2-deficient cells that were associated with reduced levels of Serine 317-phosphorylated Chk1. In aphidicolin-treated cells, there were lower levels of Serine 317-phosphorylated Chk1 when Plk2 was knocked-down. Plk2 was demonstrated to interact with Chk2, Chk1, Serine 317-phoshorylated Chk1 and p53. Thus, increased cell death observed after aphidicolin treatment and release in Plk2-deficient cells may result from both higher levels of replication stress-induced DNA damage and a dysfunctional S-phase checkpoint.

Predicting therapy response in live tumor cells isolated with the flexible micro spring array device

Cells disseminated from primary epithelial tumors into peripheral blood, called circulating tumor cells (CTCs), can be monitored to assess metastases and to provide a surrogate marker of treatment response. Here, we demonstrate how the flexible micro spring array (FMSA) device—a novel microfluidic device that enriches CTCs by two physical parameters: size and deformability—could be used in the rational development of treatment intervention and as a method to study the fundamental biology of CTCs. Cancer cells of different origins were spiked into healthy samples of donor blood to mimic blood samples of metastatic cancer patients. This spiked human blood was filtered using the FMSA device, and the recovered cells were successfully expanded in vitro and in a novel in vivo system. A series of experiments were performed to characterize these cells and to investigate the effect of chemotherapy on the resulting cultures. As few as 20 colon cancer cells in 7.5 mL blood could be isolated with the FMSA device, expanded both in vitro and in vivo and used at 25 cells per well to obtain significant and reliable chemosensitivity data. We also show that isolating a low number of viable patient CTCs and maintaining them in culture for a few weeks is possible. The isolation of viable cancer cells from human blood using the FMSA device provides a novel and realistic means for studying the biology of viable CTCs and for testing drug efficacy on these rare cells—a hypothesis that can be tested in future clinical trials.

A multiplexed marker-based algorithm for diagnosis of carcinoma of unknown primary using circulating tumor cells

Real-time, single-cell multiplex immunophenotyping of circulating tumor cells (CTCs) is hypothesized to inform diagnosis of tissue of origin in patients with carcinoma of unknown primary (CUP). In 20 to 50% of CUP patients, the primary site remains unidentified, presenting a challenge for clinicians in diagnosis and treatment. We developed a post-CellSearch CTC assay using multiplexed Q-dot or DyLight conjugated antibodies with the goal of detecting multiple markers in single cells within a CTC population. We adapted our approach to size-based CTC enrichment protocols for capturing CTCs and subsequent immunofluorescence (IF) using a minimal set of markers to predict the primary sites for common metastatic tumors. The carcinomas are characterized with cytokeratin 7 (CK7), cytokeratin 20 (CK20), thyroid transcription factor 1 (TTF-1), estrogen receptor (ER) or prostate-specific antigen (PSA. IF has been optimized in cultured tumor cells with individual antibodies, then with conjugated antibodies to form a multiplex antibody set. With IF, we evaluated antibodies specific to these 5 markers in lung, breast, colorectal, and prostate cancer cell lines and blood from metastatic prostate and breast cancer patients. This advanced technology provides a noninvasive, diagnostic blood test as an adjunct to routine tissue biopsy. Its further implementation requires prospective clinical testing.

Reduced cell death, invasive and angiogenic features conferred by BRCA1-deficiency in mammary epithelial cells transformed with H-Ras

To investigate the role of tumor suppressors BRCA1 and p53 proteins in human breast tumorigenesis, we transformed immortalized human mammary epithelial cells, MCF10A, with or without BRCA1/p53 gene-specific knockdowns. Stable knockdown of BRCA1 alone in MCF10A cells led to centrosome amplification, impaired p53 protein stability, increased sensitivity towards DNA-damaging agents, defective chromosomal condensation at mitosis and elevated protein levels of cyclin D1 and c-myc. While over-expression of mutant H-Ras transformed MCF10A cells, depletion of BRCA1 dramatically enhanced the in vivo tumorigenesis that was associated with higher levels of VEGF, enhanced vascularization and less apoptosis in the BRCA1-deficient Ras-transformed tumors. The Ras-transformed BRCA1-deficient tumors exhibited features of the epithelial-to-mesenchymal transition, appeared to secrete matrix metalloproteases as visualized by in vivo bio-imaging of tumors using fluorescent probe MMP680, and were locally metastatic to lymph nodes. Our results suggest that loss of BRCA1 pathway function may contribute to the aggressiveness of Ras-MAPK driven human breast cancer with associated increase in levels of cyclin D1 and c-myc, enhanced MAPK activity, angiogenic potential & invasiveness. This mammary xenograft tumor model may be useful as a tool to understand human breast tumor angiogenesis and metastasis, as well as to test candidate therapeutics.

Circulating tumor cells: silent predictors of metastasis

Circulating tumor cells (CTCs) were added to the arsenal of clinical testing in 2004 for three cancer types: metastatic breast, prostate, and colorectal cancer. CTCs were found to be an independent prognostic indicator of survival for these three diseases. Multiple enrichment/isolation strategies have been developed and numerous assay applications have been performed using both single and pooled captured/enriched CTCs. We have reviewed the isolation techniques and touched on many analyses. The true utility of a CTC is that it acts as a “silent” predictor of metastatic disease. The mere presence of a single CTC is an indication that disease has spread from the primary site. Comments and suggestions have been set forth for CTCs and cell-free DNA to be used as a screening panel for the early detection of disease recurrence and metastatic spread, providing the opportunity for early intervention with curative intent to treat metastatic disease.

Abstract 382: Identification and characterization of circulating tumor cells with multiplexed Quantum Dot conjugated antibodies

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA As non-invasive real-time liquid biopsy, circulating tumor cell (CTC) detection and characterization is one of the most active areas in translational cancer research, with 627 clinical trials having that include analysis of CTCs as a biomarker. We used breast cancer cells, prostate cancer cells and melanoma cells spiked into normal donor whole blood and captured with high porosity micro-filters followed by immunofluorescence staining. DAPI, CD45 conjugated with QDot525, CK8/18/19 conjugated with QDot605 and high molecular weight-melanoma associated antigen (HMW-MAA) conjugated with QDot705 were stained to identify CTCs. ER conjugated with QDot800 and PSA conjugated with QDot655 were stained to characterize CTCs. The CTC recovery rate was compared with the report from the US Food and Drug Administration- approved CellSearch® system (Veridex). Multiplexed tumor marker staining results were compared with single staining with fluorescently-labeled secondary antibody. Our study demonstrates that CTC multiplexed analysis has the potential for further genetic characterization of CTCs and offers potential insights into therapeutic targeting and resistance mechanisms. Citation Format: Lanlan Zhou, Elizabeth M. Matthew, David T. Dicker, Wafik S. El-Deiry. Identification and characterization of circulating tumor cells with multiplexed Quantum Dot conjugated antibodies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 382. doi:10.1158/1538-7445.AM2015-382

Plk2 Loss Commonly Occurs in Colorectal Carcinomas but not Adenomas: Relationship to mTOR Signaling

Plk2 is a target of p53. Our previous studies demonstrated that with wild-type p53, Plk2 impacts mTOR signaling in the same manner as TSC1, and Plk2-deficient tumors grew larger than control. Other investigators have demonstrated that Plk2 phosphorylates mutant p53 in a positive feedback loop. We investigated Plk2’s tumor suppressor functions in relationship to mTOR signaling. Archival specimens from 12 colorectal adenocarcinomas were stained for markers including Plk2, phosphorylated mTOR (serine 2448) and ribosomal S6 (Serine 235/236). We show that Plk2 is expressed in normal colon, with a punctate staining pattern in supranuclear cytoplasm. In colorectal adenocarcinoma, Plk2 demonstrates complete or partial loss of expression. Strong expression of phosphorylated mTOR is observed in the invasive front. Phosphorylated S6 expression partially correlates with phosphorylated mTOR expression but appears more diffuse in some cases. p53 and Ki67 expression is diffuse, in the subset of cases examined. In order to determine whether Plk2 is lost prior to the development of invasive cancer, 8 colon polyps from 6 patients were evaluated for Plk2 expression. All polyps are positive for Plk2. A Cancer Genome Atlas search identified Plk2 mutations to be infrequent in colorectal adenocarcinomas. Neither Plk2 methylation (in the gene body) nor copy number variations correlated with changes in mRNA expression levels. Loss of Plk2 expression along with accentuated expression of phosphorylated mTOR and phosphorylated S6 at the invasive front in some colorectal carcinomas is consistent with previous findings that an interaction between Plk2 and TSC1 / mTOR signaling molecules plays a role in tumor suppression. Plk2 protein expression is lost at the same stage in colorectal carcinogenesis as p53. The p53 dependence of Plk2 loss and tumor suppressor function in relationship to mTOR signaling may have therapeutic implications.

Abstract LB-023: Plk2 loss occurs commonly in colorectal carcinomas but not in adenomas

Plk2 is a target of p53. Our previous studies demonstrated that in the wild-type p53 context, Plk2 impacts mTOR signaling in the same manner as TSC1, and human tumor cells deficient in Plk2 grew significantly larger than control tumor cells. Other investigators have demonstrated that Plk2 phosphorylates mutant p53 in a positive feedback loop. We investigated Plk29s tumor suppressor functions as they relate to mTOR signaling. Archived pathology specimens from 12 colorectal adenocarcinomas (4 early stage and 8 advanced stage) were stained for phosphorylated mTOR (serine 2448), phosphorylated ribosomal S6 (Serine 235/236), Plk2, p53, Ki67, and glucose transporter 1 (Glut1). We show that Plk2 is expressed in normal colon epithelium, with a punctate staining pattern in the supranuclear region. In colorectal adenocarcinoma, Plk2 demonstrates partial loss of expression, complete loss of expression, or disrupted expression manifested as irregular or abnormal localization. Loss of Plk2 expression is more pronounced in the invasive front in some cases. Strong expression of phosphorylated mTOR is observed in the invasive front, which is presumed to be less hypoxic. Phosphorylated S6 and Glut1 expression partially correlate with phosphorylated mTOR expression but appear more diffuse in some cases. p53 and Ki67 expression is diffuse, rather than specific to the tumor invasive front in the subset of cases examined. In order to determine at what stage in carcinogenesis Plk2 is lost, an additional 8 archived pathology specimens (tubular adenomas, sessile serrated polyps and hyperplastic polyps) from 6 patients were evaluated for Plk2 expression. All specimens were found to be positive for Plk2 expression. We conducted a TCGA search of Plk2 alterations in colorectal adenocarcinomas. Plk2 is mutated in only 8 of 498 (all p53 wild-type) cases. Neither Plk2 methylation (in the gene body not in the regulatory region CpG islands and shores) nor copy number changes correlated with mRNA expression changes and appeared to be independent of p53 status. Loss of Plk2 expression along with accentuated expression of phosphorylated mTOR, phosphorylated S6, and Glut1 at the invasive front at least in some colorectal carcinomas is consistent with previous findings that an interaction between Plk2 and TSC1 / mTOR signaling molecules plays a role in tumor suppression during hypoxic conditions. Plk2 protein expression was found to be lost at the same stage in colorectal carcinogenesis as p53 genetic loss. The p53 dependence of Plk2 loss and tumor suppressor function as they relate to mTOR signaling may have therapeutic implications. Citation Format: Elizabeth M. Matthew, Zhaohai Yang, Suraj Peri, Namrata Vijayvergia, Amriti Lulla, Eric Ross, Wafik S. El-Deiry. Plk2 loss occurs commonly in colorectal carcinomas but not in adenomas. [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 LB-023.