The role of the MAD2-TLR4-MyD88 axis in paclitaxel resistance in ovarian cancer

Despite the use of front-line anticancer drugs such as paclitaxel for ovarian cancer treatment, mortality rates have remained almost unchanged for the past three decades and the majority of patients will develop recurrent chemoresistant disease which remains largely untreatable. Overcoming chemoresistance or preventing its onset in the first instance remains one of the major challenges for ovarian cancer research. In this study, we demonstrate a key link between senescence and inflammation and how this complex network involving the biomarkers MAD2, TLR4 and MyD88 drives paclitaxel resistance in ovarian cancer. This was investigated using siRNA knockdown of MAD2, TLR4 and MyD88 in two ovarian cancer cell lines, A2780 and SKOV-3 cells and overexpression of MyD88 in A2780 cells. Interestingly, siRNA knockdown of MAD2 led to a significant increase in TLR4 gene expression, this was coupled with the development of a highly paclitaxel-resistant cell phenotype. Additionally, siRNA knockdown of MAD2 or TLR4 in the serous ovarian cell model OVCAR-3 resulted in a significant increase in TLR4 or MAD2 expression respectively. Microarray analysis of SKOV-3 cells following knockdown of TLR4 or MAD2 highlighted a number of significantly altered biological processes including EMT, complement, coagulation, proliferation and survival, ECM remodelling, olfactory receptor signalling, ErbB signalling, DNA packaging, Insulin-like growth factor signalling, ion transport and alteration of components of the cytoskeleton. Cross comparison of the microarray data sets identified 7 overlapping genes including MMP13, ACTBL2, AMTN, PLXDC2, LYZL1, CCBE1 and CKS2. These results demonstrate an important link between these biomarkers, which to our knowledge has never before been shown in ovarian cancer. In the future, we hope that triaging patients into alterative treatment groups based on the expression of these three biomarkers or therapeutic targeting of the mechanisms they are involved in will lead to improvements in patient outcome and prevent the development of chemoresistance.


Uncropped Western Blots Western Blot Results Figure 1D
Protein lysates used in Figure 1D were initially probed for GAPDH, MyD88 and TLR4 to confirm knockdown of TLR4 and MyD88 in SKOV-3 cells in our previous PLOS One publication "The MyD88+ Phenotype Is an Adverse Prognostic Factor in Epithelial Ovarian Cancer" (PMID: 24977712) before subsequent reprobing with an antibody directed against MAD2 to determine whether disruption of TLR4/MyD88 signalling influenced protein expression levels of MAD2. TLR4 protein expression in SKOV-3 cells following knockdown of MyD88 or TLR4. The blot was exposed for 140 seconds for optimum results. The band corresponding to TLR4 is indicated by the arrow. This blot represents the first two of four replicates.
TLR4 protein expression in SKOV-3 cells following knockdown of MyD88 or TLR4. The blot was exposed for 140 seconds for optimum results. The band corresponding to TLR4 is indicated by the arrow. This blot represents the third and fourth of four replicates.
GAPDH protein expression in SKOV-3 cells following knockdown of MyD88 or TLR4. The blot was exposed for 140 seconds for optimum results. The band corresponding to GAPDH is indicated by the arrow. This blot represents the third and fourth of four replicates.

MAD2 protein expression in SKOV-3 cells following knockdown of MyD88 or TLR4.
The blot was exposed for 60 seconds for optimum results. The band corresponding to MAD2 is indicated by the arrow. This blot represents the first two of four replicates.

MAD2 protein expression in SKOV-3 cells following knockdown of MyD88 or TLR4.
The blot was exposed for 60 seconds for optimum results. The band corresponding to MAD2 is indicated by the arrow. This blot represents the third and fourth of four replicates. Figure 1G TLR4 protein expression in A2780 cells following overexpression of MyD88. The blot was exposed for 60 seconds for optimum results. The band corresponding to TLR4 is indicated by the arrow.

Western Blot Results
GAPDH protein expression in A2780 cells following overexpression of MyD88. The blot was exposed for 30 seconds for optimum results. The band corresponding to GAPDH is indicated by the arrow.
MAD2 protein expression in A2780 cells following overexpression of MyD88. The blot was exposed for 60 seconds for optimum results. The band corresponding to MAD2 is indicated by the arrow.
MyD88 protein expression in A2780 cells following overexpression of MyD88. The blot was exposed for 40 seconds for optimum results. The band corresponding to MyD88 is indicated by the arrow. Figure 2B TLR4 protein expression in A2780 cells following siRNA knockdown of MAD2. The blot was exposed for 100 seconds for optimum results. The band corresponding to TLR4 is indicated by the arrow.

Western Blot Results
MAD2 protein expression in A2780 cells following siRNA knockdown of MAD2. The blot was exposed for 60 seconds for optimum results. The band corresponding to MAD2 is indicated by the arrow. GAPDH protein expression in A2780 cells following siRNA knockdown of MAD2. The blot was exposed for 100 seconds for optimum results. The band corresponding to GAPDH is indicated by the arrow.

Western Blot Results Figure 2E
Protein lysates used in Figure 2C were obtained from SKOV-3 cells in which MAD2 was knocked down using siRNA. Membranes were probed for GAPDH, MyD88 and TLR4 and MAD2 to confirm the knockdown and to determine whether disruption of MAD2 protein expression influenced TLR4/MyD88 signalling. TLR4 protein expression in SKOV-3 cells following siRNA knockdown of MAD2. The blot was exposed for 150 seconds for optimum results. The band corresponding to TLR4 is indicated by the arrow.

MAD2 protein expression in SKOV-3 cells following siRNA knockdown of MAD2.
The blot was exposed for 100 seconds for optimum results. The band corresponding to GAPDH is indicated by the arrow.
MyD88 protein expression in SKOV-3 cells following siRNA knockdown of MAD2. The blot was exposed for 160 seconds for optimum results. The band corresponding to MyD88 is indicated by the arrow.
GAPDH protein expression in SKOV-3 cells following siRNA knockdown of MAD2. The blot was exposed for 80 seconds for optimum results. The band corresponding to GAPDH is indicated by the arrow. Figure 5B HMGB1 protein expression in A2780 cells following siRNA knockdown of MAD2. The blot was exposed for 10 seconds for optimum results. Molecular weight was confirmed using the chemiluminescence molecular weight marker (M). The band corresponding to HMGB1 is indicated by the arrow.

Western Blot Results
GAPDH protein expression in A2780 cells following siRNA knockdown of MAD2. The blot was exposed for 60 seconds for optimum results. Molecular weight was confirmed using the chemiluminescence molecular weight marker (M). The band corresponding to GAPDH is indicated by the arrow.
P53 protein expression in A2780 cells following siRNA knockdown of MAD2. The blot was exposed for 60 seconds for optimum results. Molecular weight was confirmed using the chemiluminescence molecular weight marker (M). The band corresponding to P53 is indicated by the arrow.