Elsevier

Life Sciences

Volume 310, 1 December 2022, 121046
Life Sciences

Matrix metalloproteinase 2 is a target of the RAN-GTP pathway and mediates migration, invasion and metastasis in human breast cancer

https://doi.org/10.1016/j.lfs.2022.121046Get rights and content

Abstract

RAS-related nuclear protein(RAN) is a nuclear shuttle and normally regulates events in the cell cycle. When overexpressed in cultured cells, it causes increases in cell migration/invasion in vitro and its overexpression is associated with early breast cancer patient deaths in vivo. However, the underlying mechanism is unknown. The effect of RAN overexpression on potential targets MMP2, ATF3, CXCR3 was investigated by Real-Time PCR/Western blots in the triple receptor negative breast cancer(TRNBC) cell line MDA-MB231 and consequent biological effects were measured by cell adhesion, cell migration and cell invasion assays. Results showed that knockdown of RAN lead to a reduction of MMP2 and its potential regulators ATF3 and CXCR3. Moreover, knockdown of ATF3 or CXCR3 downregulated MMP2 without affecting RAN, indicating that RAN regulates MMP2 through ATF3 and CXCR3. Knockdown of RAN and MMP2 reduced cell adhesion, cell migration and cell growth in agar, whilst overexpression of MMP2 reversed the knockdown of RAN. Furthermore, immunohistochemical staining for RAN and MMP2 are positively associated with each other in the same tumour and separately with patient survival times in breast cancer specimens, suggesting that a high level of RAN may be a pre-requisite for MMP2 overexpression and metastasis. Moreover, positive immunohistochemical staining for both RAN and MMP-2 reduces further patient survival times over that for either protein separately. Our results suggest that MMP2 expression can stratify progression of breast cancers with a high and low incidence of RAN, both RAN and MMP2 in combination can be used for a more accurate patient prognosis.

Simple summary

Ran is an important regulator of normal cell growth and behaviour. We have established in cell line models of breast cancer (BC) a molecular pathway between RAN and its protein-degrading effector MMP-2 and properties related to metastasis in culture. Using immunohistochemistry (IHC) staining of primary BCs, we have shown that RAN and MMP-2 are on their own significantly associated with patient demise from metastatic BC. Moreover, when staining for MMP-2 is added to that for RAN in the primary tumours, there is a significant decrease in patient survival time over that for either protein alone. Thus a combination of staining for RAN and MMP2 is an excellent marker for poor prognosis in breast cancer.

Introduction

Worldwide, there are an estimated 1 million cases and 0.5 million deaths from breast cancer (BC) annually [1], but the underlying mechanisms that cause metastasis and ultimately death are largely unknown. One molecule RAS-related nuclear protein, RAN is overexpressed in the primary tumours of breast cancer, and its overexpression is associated with a decrease in survival times of the corresponding patients [2], [3], presumably resulting from enhanced metastasis of the primary tumours [2]. RAN itself is a small GTPase [4] which is highly conserved in eukaryotes and is essential for cell viability [4]. It is involved in various cellular processes including nucleocytoplasmic transport, mitotic spindle organization and nuclear envelope formation [5]. RAN regulates formation and organization of the microtubule network independently of its role in the nuclear-cytosol exchange of macromolecules [6] and it also appears to be a key signalling molecule regulating microtubule polymerization during cell adhesion and cell migration [6].

Recently, we have shown that when an expression vector for RAN is transfected into benign, non-invasive rat mammary (Rama 37) cells, it has the ability to transform their phenotype, producing increased cell migration and invasion in vitro and the induction of metastasis in syngeneic rats in vivo [7]. Silencing RAN using small interfering RNAs reversed the induction of this metastatic phenotype [7], [8]. However, how RAN induces these changes is unknown. Here we identify a matrix metalloproteinase (MMP), MMP2 [9], and its upstream regulators, activating transcription factor 3 (ATF3) [10], [11], [12], [13], [14], [15], [16] and C-X-C motif chemokine receptor 3 (CXCR3) [17], [18] that are targets for the RAN-induced increases in metastasis-related properties in vitro and show a signification association between immunohistochemical staining for RAN, MMP2 in the primary tumours and survival time of breast cancer patients in vivo. We also show that staining for MMP2 acts synergistically with that for RAN in their association with survival times and use staining for MMP2 and RAN in combination to obtain a more accurate prognosis for the patient.

Section snippets

Culture conditions

The TRNBC MDA-MB231, estrogen receptor-positive MCF-7,T47D breast cancer cell lines and viral packaging cell line 293T (HEK) were obtained from the American Type Culture Collection (ATCC), Manassas, VA, USA and maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10 % (v/v) fetal bovine serum (FBS), 2 mM l-glutamine, and 1 % (v/v) penicillin and streptomycin in an atmosphere of 90 % (v/v) air, 10 % (v/v) CO2. Cells were cultured in normal medium [2] until 24 h post-infection and

Knockdown of RAN results in the downregulation of MMP2 in cancer cell lines

Previously, we had shown that knockdown of RAN using potent shRNAs results in apoptosis and changes in cell properties including cell adhesion, migration and invasion in the TRNBC cell line MDA-MB231 [2], [3]. Hence the same 3 shRNAs to RAN: shRAN1, shRAN2 and shRAN3 were separately used to infect MDA-MB231 cells to produce the stable transductants MDA-MB231-shRAN1, -shRAN2 and -shRAN3 (Materials and methods). These 3 stably-transduced cell lines produced reductions in RAN mRNA of 78 %, 79 %

Discussion

Previously, we have shown that knockdown of RAN by shRNA results in reduction of in vitro cell biological properties including cell adhesion, colony formation and cell invasion [2], [3], [19], as well as in vivo metastasis [7]. Here RAN knockdown in breast cancer cells reduces MMP2 mRNA and protein levels, probably via ATF3 and CXCR3 which, in turn, results in a significant reduction in cell adhesion and colony formation in breast cancer cell lines. However, overexpression of MMP2 in RAN

Conclusion

In conclusion, we have established in cell line models of breast cancer a relationship between RAN and MMP2 and properties related to metastasis. The inclusion of MMP2 as well as RAN facilitates a more accurate prognosis and further identifies a subgroup of patients that could benefit more from conventional chemotherapy and from therapy directed against both proteins than against either one alone.

CRediT authorship contribution statement

Mohamed El-Tanani: Concept development, Experimental design and imaging, data analysis.

Yin-Fai Lee: Experimental work and imaging, data analysis.

Arwa Omar Al Khatib: Experimental work and imaging, data analysis.

Yusuf Haggag: Experimental work and imaging, data analysis.

Mark Sutherland: Experimental work and imaging, data analysis.

Shu-Dong Zhang: Experimental work and imaging, statical analysis.

Alaa A.A. Aljabali: Experimental work and imaging, data analysis.

Vijay Mishra: Experimental work and

Ethics approval and consent to participate

Human materials and data were collected in an anonymised fashion under the legally-binding, UK Human Tissue Act supervised by the UK's National Research Ethical Committee (NREC) and approved by North West REC (Ref [12]/NW/0778), protocol number UOL000889, IRAS number 107845 to Prof. P.S. Rudland.

Consent for publication

All authors consent for publication.

Funding

Funded by Al-Ahliyya Amman University, Grow MedTech and Cancer and Polio Research Fund Ltd., UK (M. El-T, A.P-H, P.S.R).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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