Gollahon Lab Projects
Jaehyung Lee: Mitotic perturbations induced by Nek2 overexpression require interaction with TRF1 in breast cancer cells.
NIMA-related kinase 2 (NEK2), a serine-threonine protein kinase, plays a role in features of mitotic progression including timing of mitotic entry, chromatin condensation, spindle organization and cytokinesis. Kinase death NEK2 mutant expression or NEK2 depleted cells lead to failure of centrosome separation while NEK2 overexpression results in premature centrosome separation. In addition, it has been revealed that telomeric repeat binding factor 1 (TRF1) interacts directly with NEK2. TRF1 not only regulates telomere length, but is also associated with cell cycle regulation. However, the interactions and correlations between NEK2 and TRF1 are far from clear. Here, we show that mitotic aberrations through NEK2 overexpression are likely to require TRF1. Our results demonstrate that NEK2 directly binds and phosphorylates TRF1 in vitro and in vivo through multiple sites on TRF1. NEK2 overexpression in breast cancer cells, MCF 7 and MDA-MB-231, results in increased numbers of centrosomes and multinucleated cells, which leads to cytokinetic failure and aneuploidization. Additionally, TRF1 depletion by siRNA prevents the occurrence of unaligned chromosomes by NEK2 overexpression during metaphase. Concurrent Nek2 overexpression and TRF1 depleted cells demonstrated ~ 2 centrosomes per cell, similar to mock plasmid and negative control siRNA transfected cells. Therefore, we propose that TRF1 is required for overexpressed NEK2 to trigger abnormal mitosis and chromosomal instability. This is demonstrated in the following model:
From this work, we were selected for the cover in the December 2013 volume of Cell Cycle.
Zhi Pan: Investigating the role of the endoplasmic reticulum in calcium regulation with regards to paclitaxel treatment.
This work involves three separate but related projects measuring changes in ER-calcium release, paclitaxel time and treatment concentrations and the role of the anti-apoptotic protein Bcl2 in regulating calcium concentrations in the cell. This is important because of implications for PCD.
Project 1. Taxol™, an important chemotherapy agent, employs apoptosis to induce cell death in breast cancer treatment. Our previous study showed that Taxol™ directly releases calcium from the endoplasmic reticulum (ER), which can promote subsequent apoptosis in breast cancer cells. Besides its effect on the ER, Taxol™ also stabilizes microtubules, causing mitotic arrest. However, the relationship between these events and apoptosis remains unclear. Our previous studies focused on the role calcium regulation plays in the relationship between mitotic arrest and apoptosis induced by Taxol™ treatment. Our results showed Taxol™ induced significant microtubule stabilization and mitotic arrest before apoptosis was observed. Pretreatment with calcium agents interfered with both processes, indicating Taxol™-induced calcium changes act as upstream signals for subsequent processes. Our results also showed that Taxol (10-6 M) induced an immediate endoplasmic reticulum (ER) calcium release, which promoted subsequent apoptosis in breast cancer cells, whereas a lower dosage (10-7 M) used an ER calcium-independent mechanism to induce significant apoptosis. In our most recent study, the dose and duration-related calcium regulation in Taxol-induced cytotoxicity was further investigated. A range of Taxol dosages (10-9 to 10-5 M) were analyzed in breast cancer cell lines. Our results suggest 1h exposure of Taxol, depending on dosage, can induce apoptosis after several hours to days without the need of a constant Taxol supply. Taxol-induced ER calcium release acts as an early signal for subsequent apoptosis. This new finding helps to elucidate the mechanism of Taxol action with potential implications for revising treatment strategies. In another study, we investigated the relationship of BCL2 to Taxol and calcium. Expression of Bcl-2 can suppress the cell’s ability to release ER calcium. This protects the cell from releasing too much ER calcium and thus inhibits the induction of subsequent apoptosis. Meanwhile, the ER calcium store also serves as an important direct target for paclitaxel at higher dosage, and Bcl-2 expression has no effect on this dose dependent action of paclitaxel. Therefore, Paclitaxel a at higher dose is capable of releasing significant ER calcium to overcome Bcl-2’s inhibitory effect and thus attenuate Bcl-2 resistance. Our results showed that the ER calcium store is a key common target for paclitaxel and Bcl-2, since both can directly change ER calcium release independent of each other. This direct ER-calcium involvement, linking the actions of paclitaxel and Bcl-2 together, helps elucidate the underlying mechanisms of the paclitaxel - Bcl-2 relationship in a novel way, outside of the previously described, indirect ER calcium regulation - through the phosphorylation of Bcl-2 by paclitaxel. Furthermore, the dosage of paclitaxel is crucial for inducing significant ER-calcium release to overcome Bcl-2-mediated apoptotic resistance. Therefore, our findings not only helps clarify the controversy regarding the efficacy of paclitaxel in the presence of Bcl-2 expression, but may also assist oncologists in optimizing the usage of paclitaxel in breast cancer treatment. More research is needed to fully understand the complex relationship between paclitaxel, calcium and Bcl-2. Our results can be summarized in the following figure and graphic abstract.
Project 2: Taxol Directly Induces Endoplasmic Reticulum-Associated Calcium Changes That Promote Apoptosis in Breast Cancer Cells.
Calcium, a key regulator of cell survival, is also important in regulating apoptosis. Although the chemotherapeutic agent Taxol employs apoptosis to induce cell death, the exact mechanism of how it induces apoptosis and the role of calcium in this process remains unclear. The main intracellular calcium storehouse, the endoplasmic reticulum, was identified as a new important gateway in apoptosis, possibly providing a target for Taxol. The goal of this study was to investigate whether calcium changes associated with the endoplasmic reticulum, were directly or indirectly generated by Taxol at clinically relevant doses, and related to Taxol-induced apoptosis in breast cancer cells. Time-lapsed imaging techniques followed by an endoplasmic reticulum-targeted construct, cameleon D1ER, were used to monitor cytosol––endoplasmic reticulum calcium dynamics in MDA-MB-468 (Bcl-2 negative) and MCF 7 (Bcl-2 positive) breast carcinoma cells. Apoptosis levels were measured with Annexin V and Propidium Iodide (PI) using flow cytometry. In both cell lines, Taxol at 2.5 uM (10-6 M) was observed to induce significant internal calcium changes, first a rapid endoplasmic reticulum calcium release and a transient cytosolic calcium increase upon Taxol addition. After several hours of Taxol treatment, the endoplasmic reticulum calcium store was gradually depleted, and a sustained cytosolic calcium elevation was observed before significant induction of apoptosis. Inhibition of these calcium changes decreased Taxol-induced apoptosis levels. In contrast, 0.2 uM Taxol (10-7 M) induced only a slight cellular calcium change, not enough to regulate apoptosis. Our findings demonstrate that endoplasmic reticulum calcium stores provide a direct target for Taxol action and are important for induction of apoptosis, independent of Bcl-2 status. Furthermore, our results show for the first time, that the role of calcium in Taxol-induced endoplasmic reticulum-mediated apoptosis is dependent on Taxol dosage.
Project 3: Paclitaxel induces apoptosis in breast cancer cells through different calcium - regulating mechanisms depending on external calcium conditions.
Previously, we reported that endoplasmic reticulum calcium stores were a direct target for paclitaxel initiation of apoptosis. Furthermore, the actions of paclitaxel attenuated Bcl-2 resistance to apoptosis through endoplasmic reticulum-mediated calcium release. To better understand the calcium-regulated mechanisms of paclitaxel-induced apoptosis in breast cancer cells, we investigated the role of extracellular calcium, specifically; whether influx of extracellular calcium contributed to and/or was necessary for paclitaxel-induced apoptosis. Our results demonstrated that paclitaxel induced extracellular calcium influx. This mobilization of extracellular calcium contributed to subsequent cytosolic calcium elevation differently, depending on dosage. Under normal extracellular calcium conditions, high dose paclitaxel induced apoptosis-promoting calcium influx, which did not occur in calcium-free conditions. In the absence of extracellular calcium an “Enhanced Calcium Efflux” mechanism in which high dose paclitaxel stimulated calcium efflux immediately, leading to dramatic cytosolic calcium decrease, was observed. In the absence of extracellular calcium, high dose paclitaxel’s stimulatory effects on capacitative calcium entry and apoptosis could not be completely restored. Thus, normal extracellular calcium concentrations are critical for high dose paclitaxel-induced apoptosis. In contrast, low dose paclitaxel mirrored controls, indicating that it occurs independent of extracellular calcium. Thus, extracellular calcium conditions only affect efficacy of high dose paclitaxel-induced apoptosis.
Kyungwoo Lee: Telomere Biology and Cancer Stem Cells
ZSCAN4 Directly Interacts with Human Rap1 in Cancer Cells Regardless of Telomerase Status.
Telomeres are repetitive sequences at the ends of chromosomes protected by DNA binding proteins of the shelterin complex that form capping structures. Through the interaction of shelterin complex-associated proteins, telomere length maintenance is regulated. Recently, the newly identified embryonic stem cell marker, Zinc finger and SCAN domain containing 4 gene (Zscan4), was shown to be a telomere-associated protein, co-localizing to the shelterin complex. Furthermore, it was shown to play an essential role in genomic stability by regulating telomere elongation. Although, it is known that Zscan4 regulates TRF2, POT1b and Rap1 expression in embryonic stem cells, the relationship and the exact mechanism of action for Zscan4-mediated telomere maintenance in cancer cells is unknown. In this study, we investigated Zscan4 expression and interactions with Rap1 in telomerase positive (HeLa, MCF7) and ALT pathway (SaOS2, U2OS) cancer cells. Through Western, pull-down, siRNA and overexpression assays we demonstrate, for the first time, that Zscan4 directly associates with Rap1 (physical association protein). Furthermore, by generating truncated versions of Zscan4, we identified its zinc finger domain as the Rap1 binding site. Using bimolecular fluorescence complementation (BIFC), we further validate this functional interaction in human cancer cells. Our results indicate that Zscan4 functions as a mediator of telomere length through its direct interaction with Rap1, possibly regulating shelterin complex-controlled telomere elongation in both telomerase positive and alternative lengthening of telomere pathways. This direct interaction between Zscan4 and Rap1 may explain how Zscan4 rapidly increases telomere length, yielding important information about the role of these proteins in telomere biology.
The following figure demonstrates the BIFC results for a telomerase positive breast cancer cell line (MCF7).
From this work, Dr. Lee proposed two possible models of Zscan4 interaction, based on telomerase status and was given the honor of having his work featured in the Cover Art for that Issue (shown below).
Dr. Lee also identified a functional relationship between ZSCAN4 and TRF1.
ZSCAN4 and TRF1: A Functionally Indirect Interaction in Cancer Cells Independent of Telomerase Activity
Lee, K. and L.S. Gollahon. (2015). Accepted for publication in Biochemical and Biophysical Research Communications (BBRC).
The purpose of this study: Recently, the newly identified embryonic stem cell marker, Zinc finger and SCAN domain containing 4 gene (ZSCAN4), which plays a key role in genomic stability by regulating telomere elongation, was shown to co-localize with TRF1 foci. This suggests that the interaction of ZSCAN4 with TRF1 functions in regulation of telomere elongation in ESC. Based on these studies, we hypothesized that ZSCAN4 binds to TRF1 in cancer cells to function in regulating telomere length. The purpose of this study was to determine whether this interaction occurred across different cell lineage-derived cancers and whether telomerase status impacted this relationship. To that end, telomerase positive cervical cancer cells (HeLa) and breast cancer cells (MCF7), and telomerase negative osteosarcoma cells (SaOs2), were analyzed for ZSCAN4 and TRF1 interactions. In this study, he demonstrated, for the first time, that ZSCAN4 indirectly interacts with TRF1 (functional association protein) in cancer cells. Furthermore, his data showed that ZSCAN4 plays an important role independent of telomere maintenance pathways (telomerase positive and ALT) or cell lineage.
Highlights from the paper:
Confocal images of BiFC results for ZSCAN4 and TRF1 protein complex interactions in MCF7, HeLa and SaOS2 cancer cells.DNA was labeled with DAPI (blue, shown in panel 1). Actin filaments were labeled with phalloidin (red, shown in panel 2). Panel 3 is the BiFC signal generated by interaction of the GFP fluorophore components based on proximity. Panel 4 represents the merged images. The images shown in panel 3 of A, B, and C are representative of BiFC analysis for the interaction between ZSCAN4 and TRF1 demonstrate the close proximity of ZSCAN4 and TRF1 in cancer cells.
Hongtao Ma - ERα Mediates Estrogen-Induced Expression of the Breast Cancer Metastasis Suppressor Gene BRMS1
Abstract: Recently, estrogen has been reported as putatively inhibiting cancer cell invasion and motility. This information is in direct contrast to the paradigm of estrogen as a tumor promoter. However, data suggests that the effects of estrogen are modulated by the receptor isoform with which it interacts. In order to gain a clearer understanding of the role of estrogen in potentially suppressing breast cancer metastasis, we investigated the regulation of estrogen and its receptor on the downstream target gene, breast cancer metastasis suppressor 1 (BRMS1) in MCF-7, SKBR3, TTU-1 and MDA-MB-231 breast cancer cells. Our results showed that estrogen increased the transcription and expression of BRMS1 in the ERα positive breast cancer cell line, MCF-7. Additionally, the ERα specific agonist PPT also induced the transcription and expression of BRMS1. However, the two remaining estrogen receptor (ER) subtype agonists had no effect on BRMS1 expression. In order to further examine the influence of ERα on BRMS1 expression, ERα expression was knocked down using siRNA (siERα). Western blot analysis showed that siERα reduced estrogen-induced and PPT-induced BRMS1 expression. In summary, this study demonstrates estrogen, via its α receptor, positively regulates the expression of BRMS1, providing new insight into a potential inhibitory effect of estrogen on metastasis suppression.
Graphical Abstract
Protein expression levels for each of the ER subtypes. Western analysis was performed for MCF-7, MDA-MB-231, TTU-1, and SKBR3 cells. A total of 40 µg of cell lysate was loaded onto 10% SDS-PAGE gels and transferred to nitrocellulose membranes (Hybond TM-C super, Amersham Biosciences). The membranes probed with monoclonal antibodies against ERα (1:1,000), ERβ (1:1,000), GPR30 (1:200) or α-tubulin (1:10,000). After washing, the membranes were incubated with goat anti-mouse HRP-conjugated secondary antibody (1:5,000) or goat anti-rabbit HRP-conjugated secondary antibody (1:5,000), for 1 h at room temperature. HRP activity was detected using an enhanced chemiluminescence substrate (Pierce) and exposed on blue X-ray film (Phenix Research Products). Protein was quantitated using Image J.
Western blot analysis summarizing siERα results for BRMS1 expression. MCF-7 cells were either transfected with siERα for 48 h and then treated with E2 for 24 h (A) or PPT for 16 h (B). siControl cells were incubated in the same volume of transfection reagent without siERα. ERα and BRMS1 expression was determined by Western blot (α-tubulin as control). 20 μg of the total protein was loaded for each lane. The bar graph summarizes BRMS1 protein normalized to α-tubulin from the same lane in three independent experiments. Values with error bars were the average of three independent experiments ± SD. * indicates statistic significance at the p < 0.05 level compared to siControl E2 or PPT treatment.
A proposed model for the relationship between ERα and BRMS1. (A) The BRMS1 5’ upstream region shows several putative regulatory elements, one of them is the binding site for CREB. ERα may regulate gene expression as a co-regulator on other promoter sites. ERα has been shown to bind c-Jun/ATF proteins, which bind to the CRE of the cyclin D1 promoter, inducing its expression. This suggests that ERα may induce BRMS1 expression by binding transcriptional complexes on the CRE in the promoter region of BRMS1. (B) It has been shown that ERα upregulates gene transcription by binding the AP-1 complex on AP-1 response elements. This information, taken together, suggests that ERα may induce BRMS1 expression through binding transcriptional complexes as a co-activator on other promoter sites (e.g. CRE and AP-1).
