|dc.description.abstract||Breast cancer is a complex heterogenous disease with distinct molecular subtypes and metabolic behaviour, disparate responses to therapies, and considerable differences in the overall survival of the patients. Insights into the biological heterogeneity of the disease has led to the development of therapeutic strategies for the effective treatment of breast cancer. The current treatment options include hormone therapy for estrogen positive (ER-positive); anti-HER2 antibody (trastuzumab) therapy for human epidermal growth factor receptor 2 (HER2)-amplified, and general chemotherapy for triple-negative (TN) breast cancers. Unfortunately, therapeutic outcomes remain poor due to inherent or acquired resistance of the cells to the treatments and the toxicity associated with therapy. Metabolic adaptation of breast cancer cells is considered to play a crucial role in enabling the cells to become resistant to therapy and acquire metastatic potential. Bioenergetic alteration (the Warburg effect) is fundamental to all forms of cancer and is regarded as one of the hallmarks. The Warburg effect is characterized by the presence of glycolytic phenotypes within the tumor tissue. Like most cancers, an increase in lactate production observed in breast cancers may be related to the upregulation of lactate dehydrogenase (LDH) enzyme that catalyses the conversion of pyruvate to lactate. Recently, human RasGAP -SH3 domain binding protein (G3BP), an RNA binding protein, was shown to interact with the mRNA of mitochondrial H+-ATP synthase subunit β (β-F1-ATPase), an enzyme that the mediates ATP production through oxidative phosphorylation in mitochondria. That study implicated the role of G3BP in the glycolytic phenotype observed in cancers. Indeed, G3BP is overexpressed in all forms of cancers including breast cancer. The focus of this study was to investigate the potential relevance of G3BP-1 as a breast cancer biomarker and targeting G3BP-1 by lapatinib (Lap) to inhibit the morphological adaptation and cell migration of SKBR3 breast cancer cell line.
Whilst, much of the signaling mechanisms have been elucidated, the role of G3BP in the glycolytic switch remains elusive. The first part of this study investigated the potential role of G3BP in the regulation of lactate dehydrogenase A (LDH-A), a key regulator of the glycolytic shift in cancer cells.
Thus far, research has not addressed this regulation. In order to achieve this, small interfering RNA (siRNA)-mediated knockdown of G3BP-1 and G3BP-2 (isoforms of G3BP) was performed in MDA-MB-435 breast cancer cell line. Following the knockdown of G3BP, changes in the expression of LDH-A at the transcriptional and translational level were evaluated. The results showed that G3BP-1 (but not G3BP-2) was implicated in the translational regulation of LDH-A. Whilst, depletion of endogenous G3BP-1 resulted in a significant downregulation of LDH-A protein, silencing of G3BP-2 had no effect on the translational regulation of LDH-A. Furthermore, depletion of G3BP-1 and G3BP-2 had no effect on the endogenous mRNA levels of LDH-A. These findings suggest the role of G3BP-1 (but not G3BP-2) in regulating the bioenergetic phenotype of MDA-MB-435 breast cancer cells.
Furthermore, the effect of Lap on the protein levels of G3BP-1 and LDH-A was explored in a panel of breast cancer cell lines including SKBR3 (HER2-positive), MDA-MB-231 (triple-negative), and T47D (ER-positive/PR-positive). The data indicated that Lap significantly downregulated the protein levels of G3BP-1 and LDH-A, independent of HER2 status. There was a significant reduction of G3BP-1 and LDH-A protein levels in Lap-sensitive SKBR3, and Lap-insensitive MDA-MB-231 and T47D cell lines. Furthermore, the findings also indicate that irrespective of the breast cancer subtypes (based on the receptor status), G3BP-1 regulates the protein levels of LDH-A. This argument supports the data obtained from the translational regulation of LDH-A upon G3BP-1 knockdown in MDA-MB-435 breast cancer cell line. Hence these findings have updated the role of G3BP-1 in the glycolytic shift observed in cancers, in this context by regulating the expression of LDH-A protein in a panel of breast cancer cell lines. On a side note, the endogenous levels of G3BP-1 and LDH-A were found to be significantly higher in the breast cancer cell lines used in the study. This observation may justify the potential relevance of G3BP-1 and LDH-A as breast cancer biomarkers.
Since its initial identification as a RasGAP binding protein, several studies have implicated the role of G3BP in signaling pathways including Ras signaling, HER2 signaling, NF-κB signaling, and c-myc mRNA turnover. Indeed, these pathways are often derailed in cancer. In addition, findings in this study have reported significantly higher levels of endogenous G3BP-1 in breast cancer cell lines.
Based on these evidences, G3BP-1 may be a relevant breast cancer biomarker. Therefore, strategies to target G3BP may provide therapeutic advantages over currently available standard treatment modalities to induce sensitivity of resistant breast cancers to anti-G3BP therapy.
In the past, the mechanism of action of some anticancer drugs, including resveratrol and epigallocatechin gallate (EGCG), was elucidated by their direct interaction with the recombinant G3BP. Hence, the strategy of recombinant G3BP production was adopted in this study to identify potential anticancer agents that interact with G3BP. Since, Lap downregulated the protein levels of G3BP-1 in breast cancer cell lines (as reported in this study), protein thermal shift assays (PTS) were performed to investigate a potential interaction of recombinant G3BP-1 with Lap.
The first part of study in Chapter 4 concerns construct design, expression, and purification of the full length G3BP-1 and G3BP-2 respectively. This was achieved by inserting the respective gene sequences into the bacterial pRSETC vector with N-terminal histidine His(6)-tag, transformation into competent BL21 (DE3) E.coli cells, isopropyl-β-D-thiogalactoside (IPTG) induction, and nickel-nitrilotriacetic acid (Ni-NTA) purification of His-tagged recombinant G3BPs. The results showed that purified full length G3BP-1 and G3BP-2 corresponded to molecular weights of 65-kDa and 68-kDa respectively. The identity of the proteins was also confirmed by immunoblot analysis using protein-specific antibodies for G3BP-1 and G3BP-2. Upon testing for the expression as well as the recovery of the protein in the soluble fraction, it was observed that G3BP-1 was more efficiently recovered at 25oC compared to G3BP-2. The observed difference in the protein recovery may be attributed to the divergent primary structure of these related, yet distinct proteins. Differences in the composition of the catalytic domains between G3BP-1 and G3BP-2 may have an impact on the recovery of the proteins in the soluble fraction. Nevertheless, his(6)-G3BP-1 was purified using Ni-NTA resin with high purity and a yield of 6mg/l culture.
In the next step, the effect of Lap on thermal stabilization of purified G3BP-1 protein was studied using PTS assay. The results showed that the melting temperature (tm) of G3BP-1 in the presence of Lap was higher than for the control. Such thermal stabilization was greatest at the 4:1 stochiometric ratio of Lap to G3BP-1, with an observed thermal shift (Δtm) of ~≤1oC at 15μM of G3BP-1. To distinguish the effect of Lap on thermal stabilization of G3BP-1, Lap was compared to 5-flourouracil (5-FU), a non-ATP-interacting thymidylate synthase inhibitor. G3BP-1 thermal stability in the presence of 5-FU was measured under the same experimental conditions of 4:1 stochiometric ratio of the compound to the protein. In contrast to the earlier observation, the results showed that 5-FU did not induce thermal stabilization of G3BP-1. These findings confirmed that the observed thermal stabilization of G3BP-1 was specific to Lap. In addition, a slightly higher shift in the tm of G3BP-1 may be indicative of a weak interaction of Lap with the protein.
Metastasis is a process that involves migration of cancer cells from the primary tumor site to the distant organs. Approximately 90% of the breast cancer related mortalities have been attributed to the metastatic spread of the disease. Like other cancers, breast cancer metastasis is associated with poor prognosis. Of all the subtypes of breast cancers, HER2-amplified and triple-negative (TN) forms are known to be highly metastatic in nature. Currently, the ability to accurately predict the risk for metastatic potential poses a substantial challenge for clinical management of the disease. Interestingly, adaptive responses are induced in cancer cells following low doses of some chemotherapeutic agents.
In chapter 5, it was observed that 0.1μM doxorubicin (Dox) treatment increased the cell viability of SKBR3 breast cancer cell line. Such an increase in cell viability following Dox treatment was not observed in MDA-MB-231, T47D and HMF cell lines. Furthermore, Dox treatment also induced migratory phenotypes in SKBR3 cells. The observations based on the immunofluorescence images of the Dox treated cells revealed that there was a significant increase in the prominent F-actin filaments and the mitochondrial spread as compared to the corresponding controls.
In line with these observations, an in vitro scratch wound assay revealed that Dox treated cells showed an enhanced ability to migrate into the scratch wound area as compared to the control cells. However, a combination dose of 0.1μM Dox+5μM Lap not only decreased the viability of SKBR3 cells, but also effectively suppressed the migratory phenotype compared to Dox treated cells. In addition, cells that received the combination therapy showed a significant decrease in the percentage of wound closure compared with the corresponding Dox treated controls. Furthermore, Western blot analysis indicated that SKBR3 cells treated with Lap in combination with Dox showed a significant reduction in the protein levels of G3BP-1 and LDH-A when compared to either vehicle control or Dox treatment alone. Collectively, Lap treatment inhibited Dox-induced migratory phenotypes of SKBR3 breast cancer cell line with a significant downregulation of endogenous G3BP-1 and LDH-A proteins in these cells.||