The matrix metalloproteinase 7 (MMP7) links Hsp90 chaperone with acquired drug resistance and tumor metastasis

Abstract Background Cancer emergence is associated with a series of cellular transformations that include acquired drug resistance followed by tumor metastasis. Matrix metalloproteinases (MMPs) and Hsp90 chaperone are implicated in tumor progression, however, they are not studied in the context of drug resistance. Aims In the present study, we aimed at understanding the cross‐talk between acquired drug resistance and tumor progression, linking MMP7 and Hsp90. Methods and results We have developed an in vitro model system for acquired drug resistance and studied the correlation between MMP7 and Hsp90. We demonstrate that enhanced drug efflux activity correlates with the induced expression and activity of MMP7, and enhanced metastatic potential of cells, however, in Hsp90‐dependent manner. The MMP7 overexpression alone could enhance the drug efflux activity marginally, and metastasis significantly. However, challenging these cells with 17AAG has significantly increased the drug efflux activity and, in contrast, decreased the metastatic potential. Evaluating our in vitro findings in mice xenografts revealed that MMP7 overexpression facilitates altered homing properties. However, these cells, in response to 17AAG treatment, exhibited increased localized tumor growth but decreased tumor metastasis. Conclusion We demonstrated a cross‐talk between Hsp90 and MMP7 in regulating the acquired drug resistance and tumor progression. Our findings provide novel insights on targeting drug resistant‐tumors.

The multidrug resistance facilitates the efflux of drugs and toxic compounds by the cells. 6 The acquired drug resistance allows tumor cells to overcome therapeutic insults, thus promotes tumor aggression.
The drug resistance majorly mediated by P-glycoprotein (P-gp) and a subset of multidrug resistance proteins (MRPs) both belong to the ATPbinding cassette (ABC) family of transporters. 7,8 Linking tumor evolution with drug resistance and metastasis has been a long-standing debate.
However, it is agreed that both the cellular processes coordinately function to promote tumor survival, spread, and progression. Till date, no direct correlation is being demonstrated between drug resistance with tumor metastasis by any of these studies. [9][10][11] The cancer chaperone, Hsp90 has been implicated in tumor progression, especially in promoting the proliferative potential of cells through stabilizing the functions of mutated kinases. 12 Hsp90 also implicated in tumor cell migration, invasion, and angiogenesis. [13][14][15] While the small molecular weight heat shock proteins (Hsps) such as Hsp27 and Hsp70 are implicated in drug resistance, their role in tumor metastasis is less understood. 16,17 However, Hsp90 is extensively studied for its role in tumor metastasis, but not in drug resistance. 18,19 Hsp90 contributes to tumor metastasis through stabilizing the functions of MMPs such as MMP2, and MMP9. 20,21 Both MMP2 and MMP9 are extensively studied for cancer metastasis in a large variety of experimental as well as clinical cancer models, therefore, inhibiting, MMPs is suggested to combat cancer. 22 Earlier, we demonstrated that Hsp90 plays a role in the acquired multidrug resistance of cancer cells. 23 By extrapolating these studies, we asked whether there is any cross-talk between Hsp90 and MMP7 in regulating the drug resistance and tumor metastasis. The MMP7 can act upstream of MMP2 and MMP9, and similar to Hsp90 also overexpressed in highly metastatic cells. 24 While studying the effects of Hsp90 inhibition against the drug-resistant tumor cells, we have come across MMP7 interference with the therapeutic response mediated by Hsp90 inhibitor, 17AAG. Therefore, we have examined the functional consequence of MMP7 expression in regulating the drug resistance and tumor metastasis.
Our findings provide novel insights linking Hsp90 with MMP7 in regulating these two mechanisms, thus may have clinical implications.

| Cell culture maintenance and drug treatments
The KB cells (ATCC-CCL-17), HeLa derivative, were procured from American Type Culture Collection (ATCC), authenticated using short tandem repeat (STR) DNA profiling at CSIR-CCMB and tested for contamination from mycoplasma (DAPI staining), bacteria (medium turbidity), and yeast (appearance of bead-like outgrowth in the culture plate correlating with turbidity) before using. Cells maintained in DMEM (GIBCO, #12100-061) containing 10% FBS (GIBCO, #16000-044) in the presence of penicillin, streptomycin, and kanamycin at 37 C in a humidified incubator with 5% CO 2 supply. The 2D culture cells were grown on a plastic surface (NUNC), and for 3D cultures, the plastic surface coated with matrigel (BD Biosciences, #354234) before plating the cells. The cells (0.2 × 10 6 ) grown in a 6-well culture dishes (Thermo Scientific, #140675) and a final concentration of 2.0 μM 17AAG (#ant-agl-25, Invivogen) was used for treatments based on our earlier studies. 25 Quercetin at a final concentration of 10 μM (#Q4951-10G, Sigma-Aldrich) was used to inhibit the stress response. 26 The drug-resistant cells were prepared by treating KB Parental cells with lower to higher concentrations of vinblastine (0.5-2.0 μM, Sigma-Aldrich, V1377-5MG), and its combination with cisplatin (0.5-2.0 μM, Sigma-Aldrich, C2210000) for 60 passages and named KBvb and KBvbcp (drug-resistant), respectively. 23

| Doubling time calculation
The doubling time is the time required for cells to double in number.

| RNA isolation, reverse transcriptase polymerase chain reaction (RT-PCR)
The total RNA isolated from the cells using Trizol (Invitrogen, #15596018). First strand cDNA was synthesized from 1 μg of total RNA using the Primescript cDNA synthesis kit (Takara Clontech,

| Quantitative PCR analysis
The quantitative PCR was performed from the cDNA obtained in Cells stained with PI containing RNase A (10 μg/mL, Thermo Scientific, # ENO531) were analyzed by FACS.

| Immunoblot analysis
Cells were lysed into RIPA buffer (Radioimmunoprecipitation assay buffer, pH 8.0) and incubated at 4 C for 1 hour with gentle agitation.

| Gelatin zymography
To the 30 μL sample prepared from the conditioned medium,

| Transfection by Lipofectamine 3000
Transfection of MMP7 OE and MMP7 KD plasmids were performed as per the manufacturers' instruction. In brief, the 4 × 10 5 cells per well were plated into six-well culture plate, after 5 hours the Lipofectamine and 2 μg plasmid DNA were mixed in serum-free medium and incubated at room temperature for 10 minutes. The media was removed from the cells and 600 μL fresh complete medium was added. The lipid-DNA complex was added drop by drop and mixed thoroughly by shaking the plates. Cells were incubated at standard culture conditions for 5 hours, and the media was replaced with complete medium. After 24 hours, particular antibiotics were added for the stable selection and continued for 21 days.

| Angiogenesis assay
The angiogenesis assay or the tube formation ability of cells is deter-

| Tumor invasion assay
The invasive potential of cells was examined by transwell (Corning,

| Animal ethics statement and mice xenograft models
This study was carried out as per the guidelines and recommendations of the National Institute of Health (NIH

| Statistical analysis
The data represented are from three independent experiments mean ± SD. The statistical significance is calculated from Student's t-test for 3 | RESULTS

| The drug-resistant cells display induced MMP expression and heat shock response upon Hsp90 inhibition
The MMPs play a central role in tumor evolution, primarily through tumor metastasis. 29 Further, MMP7 is considered for the prognostic assessment as well as for the therapeutic outcome. 30 Therefore, to understand whether acquired drug-resistance correlates with MMPs expression, we have examined the expression levels of a spectrum of major MMPs in the drug-resistant cells (data not shown). We observed a significant increase in MMP7 expression in response to 17AAG treatment both in the Parental as well as in drug-resistant (KBvb) phenotypes ( Figure 1A). However, we could not see any alterations in cellular MMP7 protein levels suggesting that activated protease might have secreted out to serve its extracellular functions ( Figures 1B and S3). In agreement with our earlier findings, we observed enhanced drug efflux activity in response to 17AAG treatment ( Figure 1C). Further, we also demonstrated that P-glycoprotein (P-gp) majorly contributes to the acquired drug resistance, therefore, in the present study, the drug efflux activity that we mentioned is pertinent to P-gp activity. 23 Since induced stress response is grossly implicated in cellular adaptations that make tumor cells to evolve, 31 we have examined the heat shock response or stress response through induced Hsp synthesis in presence and absence of quercetin, a drug that interferes with the intracellular stress response. 26 In agreement with previous reports that Hsp90 inhibitors can induce stress response via the feedback loop mechanism by inducing the transcription of heat shock genes, 23 we have observed induced expressions of Hsp70, Hsp90α, and Hsp90β in both Parental and KBvb cells in response to 17AAG treatment. However, quercetin could decrease the basal Hsp-gene transcription; however, it did not interfere with Hsp-gene transcription triggered by 17AAG treatment (Figure 1D-F). 23 Interestingly, in agreement with the previous study, quercetin treatment itself triggered the drug efflux activity ( Figure 1G) 23 and did not interfere with MMP7 expression ( Figure 1H). These studies have confirmed that MMP7 expression is HSF1 independent ( Figure 1I). Since we speculated that  Figure 1K).

|
The MMP7 OE potentiates the drug efflux activity and metastatic potential of tumor cells, however, is more sensitive to Hsp90 inhibition  expressions showed a marginal increase in response to 17AAG treatment ( Figure 3D,E). The expressions of VIM and CDH2 were unaltered F I G U R E 3 MMP7 overexpression does not correlate with acquired stemness or epithelial to mesenchymal transition (EMT). A and B, RT-PCR analysis of hypoxia response factors, HIF1-α and VEGF expressions. Note that 17AAG treatment decreasing the expression levels of HIF1-α, but not VEGF. C-E, RT-PCR analysis of epithelial markers, MUC1, CK18, and TJP1 expressions. Note MUC1 expression is being decreased by 17AAG treatment in MMP7 OE cells. F-I, RT PCR analysis of mesenchymal markers, SNAIL, TWIST, VIM, and CDH2 expressions. Note that 17AAG treatment increased the expression of SNAIL. J and K, RT-PCR analysis of stem markers CD24 and CD44 expressions. Note that 17AAG treatment increasing the expressions of CD24 and CD44. L and M, RT-PCR analysis of pluripotency markers Hes-1 and Hey-1 expressions. Note decreased pluripotency with 17AAG treatments. N-P, RT-PCR analysis of MDR1, Hsp90α, and Hsp90β. The significance values calculated from Student's t-test and represented as, * P < 0.05, ** P < 0.01 either by MMP7 OE or by 17AAG treatment (Figure 3H,I). Whereas SNAIL expression was increased by 17AAG and TWIST expression was increased by MMP7 overexpression (Figure 3F,G).
Since there are no alterations in either epithelial or mesenchymal gene expressions, we want to examine their stem and pluripotent status.
MMPs are implicated in cell fate decisions, 35 hence we examined for the

| MMP7 KD decreases the drug efflux activity of tumor cells
The heat shock protein expression is correlated with both the aggressiveness of the tumor, which can be mediated by MMPs. 36   Our results demonstrate that MMP7 can influence the homing properties of tumor cells, and 17AAG treatment inhibits ( Figure 5C,D). The tumor volume comparison between different s.c. injected animals before sacrificing indicates that 17AAG treatment significantly increases the s.c. tumor volume in MMP7 OE cells ( Figure 5E). The number of metastatic foci were found to be maximum in lung, kidney, and spleen of mice injected with MMP7 OE cells, however, was inhibited by 17AAG treatment ( Figure 5F).

| DISCUSSION
The tumor spread is inhibited if tumor cells fail to reach new locations through their metastatic potential. 40