文献

Cell Cycle 11:5, 865-870; March 1, 2012; ? 2012 Landes Bioscience

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Key words: metformin, breast cancer, mitochondria, MHC-I, immunosurveil-lance, HER2

Submitted: 12/15/12

Accepted: 01/03/12

https://www.wendangku.net/doc/2317147973.html,/10.4161/cc.11.5.19252 *Correspondence to: Javier A. Menendez; Email: jmenendez@https://www.wendangku.net/doc/2317147973.html,; jmenendez@ https://www.wendangku.net/doc/2317147973.html, A ctive avoidance by tumor ce lls of

attack and e limination by immune cells is an emerging cancer hallmark that is achie ve d primarily by de cre asing the le ve ls of major histocompatibility com-plex class I (MHC-I) at the cancer cells’ surface. Deficiencies in MHC-I antigen-re stricte d immunosurve illance may be inte rtwine d with an alte re d, Warburg-lik e canc e r c e ll-intrinsic m e tabolism, anothe r e me rging hallmark of cance r that involve s a switch from mitochon-drial re spiration to glycolysis to e ffi-cie ntly support large-scale biosynthe tic programs that are re quire d for active cell proliferation. We recently envisioned that int rv ntion strat gi s aim d at re ve rsing the bioe ne rge tic signature of cancer cells (e.g., the antidiabetic bigua-nide metformin) should correct oncogene (e.g., HER2)-driven MHC-I defects, thus pre ve nting immune e scape of oncoge ne transformants. First, we e xplore d how m tformin tr atm nt impact d mito-chondrial bioge ne sis in culture d bre ast cance r ce lls ove re xpre ssing the me m-brane tyrosine kinase re ce ptor HER2, the be st-characte rize d downre gulator of MHC-I. Metformin exposure was found to dose-dependently increase the expres-sion levels of cytochrome c oxidase I and mitochondrial succinate de hydroge nase, which are encoded by mitochondrial and nucle ar DNA, re spe ctive ly. Se cond, we e xplore d whe the r me tformin-e nhance d mitochondrial bioge ne sis might signifi-cantly alter the MHC-I status in bre ast

Metformin rescues cell surface major histocompatibility complex class I (MHC-I) deficiency caused by oncogenic transformation

Cristina Oliveras-Ferraros,1,2,? Sílvia Cufí,1,2,? Alejandro Vazquez-Martin,1,2 Octavio J. Menendez,1 Joaquim Bosch-Barrera,2,3

Bego?a Martin-Castillo,2,4 Jorge Joven5 and Javier A. Menendez1,2,*

1Translational Research Laboratory; 2Girona Biomedical Research Institute; 3Unit of Clinical Research; Medical Oncology; Catalan Institute of Oncology; Girona; 4Centre de Recerca Biomèdica; Hospital Universitari Sant Joan de Reus; 5Institut d’Investigaciò Sanitària Pere Virgili; Universitat Rovira i Virgili; Reus, Catalonia Spain

?These authors contributed equally to this work.

carcinoma ce lls. MHC-I e xpre ssion, as

assessed by flow cytometry using an anti-

HLA-ABC monoclonal antibody, was

fully re store d (up to ~25-fold upre gula-

tion) in MHC-I-ne gative HER2 ge ne-

amplified carcinoma cells. These findings

may help delineate a previously unrecog-

nize d me chanism through which me t-

formin (and metformin-like drugs) may

e nable a cance r patie nt’s own immune

system to mount an efficient anti-metas-

tasis re sponse that can pre ve nt or de lay

dise ase re curre nce. Re store d antige nic-

ity and immunoge nicity of tumor ce lls

may represent a previously unrecognized

primary mode of action unde rlying the

cancer-preventive effects of metformin.

Defects in major histocompatibility com-

plex class I (MHC-I) antigen presentation

are found in most types of human cancers,

with an incidence ranging from approxi-

mately 30–80% of cases depending on the

tumor type.1,2 While MHC-I defects are

already present in early malignant lesions,

loss or downregulation of MHC-I are

more frequently associated with invasive/

metastatic disease progression and poorer

prognoses in ovarian, colorectal and breast

carcinomas.3-5 Accordingly, MHC-I mol-

ecules are significantly downregulated

in migrating cancer cells in vitro and in

invading cancer cells in vivo,6 and less

differentiated cancer stem cells (CSCs)

seem to be associated with weaker expres-

sion of MHC-I molecules.7 Cumulatively,

these findings strongly suggest that low

of cultured breast cancer cells has been shown to decrease both the activity and expression levels of the HER2 oncogene in a dose-dependent manner. At low, micro-molar doses, HER2 tyrosine kinase activ-ity is blocked, but HER2 protein levels are not affected; conversely, at high, mil-limolar doses, the expression of the HER2 protein is downregulated, independent of the molecular mechanism that contrib-utes to the overexpression of HER2.29-32 Thus, we explored for the first time how metformin treatment impacts mitochon-drial biogenesis and MHC-I expression in cultured breast cancer cells with gene amplification or transcriptional activation of the human HER2 oncogene. We chose to investigate metformin’s effects on the restoration of cell surface MHC-I expres-sion in HER2-overexpressing cancer cells, because the expression of the HER2 oncogene is one of the best-characterized mechanisms through which human car-cinomas evade host immune surveillance. There is an inverse correlation between HER2 and MHC-I expression in murine and human tumor models, which results in reduced sensitivity to the lysis of tumor cells by CTLs.33-36 Moreover, HER2-mediated downregulation of MHC-I anti-gen processing has been shown to prevent CTL-mediated tumor recognition upon DNA vaccination in HLA-A2 transgenic mice;37 these data suggest that HER2-overexpressing tumors might escape from CTLs specific for tumor antigens, and that vaccines targeting HER2 should aim to induce an integrated immune response to overcome HER2-induced tumor resistance to specific T-cell effector mechanisms.

To test our hypothesis, we employed a commercially available MitoBiogenesis TM In-Cell ELISA that uses quantitative immunocytochemistry to measure drug-induced effects on mitochondrial biogen-esis in cultured cells (Fig. 1B ). For these in vitro studies, we utilized the SKBR3 breast cancer cell line, a widely employed model characterized by spontaneous HER2 gene amplification, HER2 recep-tor protein overexpression (~1,000 ng HER2 mgprotein -1) and HER2 depen-dency for cell proliferation and survival.38 We seeded SKBR3 cells in triplicate at 5,000 cells/well and allowed them to

antigen presentation. Interferon treat-ment consistently rescues MHC-I defects in human cancer cell lines and improves CTL-mediated lysis.17 Although histone methylase or de-acetylase inhibitors and interferons are all safe and well-tolerated as monotherapies and can be tested in com-bination with different forms of immu-notherapy, accumulation of acetylated histones in normal tissues may induce toxicity. Furthermore, these treatments can also lead to unwanted changes in gene expression profiles, and tumors can develop permanent resistance to epigene-tic modifications or interferon-based treat-ments. The above-mentioned limitations underscore our urgent need to describe and develop novel molecular approaches aimed at counteracting MHC-I defects in tumors.

Charni et al. have recently provided indirect evidence that inhibitors of gly-colysis and/or enhancers of mitochondrial respiration could serve as previously unex-plored treatments for rescuing MHC-I deficiency in tumors. Active avoidance by cancer cells from attack and elimina-tion by immune cells is an emerging hall-mark of cancer that is achieved primarily through decreasing the levels of MHC-I at the cancer cell surface.19-21 Charni’s study showed for the first time that this mechanism is intertwined with an altered, Warburg-like cancer cell-intrinsic metabolism, another emerging hallmark of cancer; this phenomenon involves a switch from mitochondrial respiration to glycolysis, a molecular mode that effi-ciently supports large-scale biosynthetic programs that are required for active cell proliferation.22-25 In their hands, culture conditions that forced mitochondrial res-piration significantly upregulated MHC-I transcription and protein levels at the cell surface of leukemia cells. Conversely, leu-kemia cells utilizing glycolysis or lacking a functional mitochondrial respiration chain displayed a significant decrease in MHC-I levels at the cell surface. We recently hypothesized that treatment with the anti-diabetic biguanide drug met-formin might rescue MHC-I deficiency because of metformin’s ability to alter the glucose and lactate/pyruvate fluxes while stimulating mitochondrial biogen-esis (Fig. 1A ).26-28 Metformin treatment efficiency in MHC-I-related antigen processing and presentation preferen-tially occurs in the most biologically aggressive subset of cancer cells within heterogeneous tumor populations. As tumor-reactive cytotoxic T lymphocytes (CTLs) lyse tumor cells through the rec-ognition of tumor antigens on MHC-I, the reduced levels of MHC-I may result in decreased sensitivity to CTL-mediated lysis and thereby help tumor cells to evade the classical T cell-dependent immune surveillance.8 It could be argued that loss of MHC-I expression merely reflects the genetic instability of tumor cells; how-ever, MHC-I processing defects represent a real mechanism by which tumor cells escape from immune pressure. Recent studies have confirmed that the difference between regressing and progressing metas-tasis during immunotherapy correlates

with the failure to restore MHC-I expres-sion in progressing lesions.9,10 Hence, if this molecular alteration in tumor cells is reversible by immunotherapy, the expres-sion of MHC-I will be upregulated, and the lesion will be successfully recognized and rejected.11-13

The molecular mechanisms underly-ing MHC-I antigen processing defects can be categorized as reversible (or “soft” defects) or irreversible (or “hard” defects). Irreversible defects commonly result from structural defects in the MHC-I gene, such as loss of heterozygosity (LOH) or point mutations, which are frequently found in certain cancer types.2,14 Reversible defects depend on the repression of MHC-I gene expression, which is often exerted via histone modifications and can be regu-lated by well-known cancer-related genes, including p53 and HER2.15,16 The fact that MHC-I antigen presentation is not usually controlled by structural altera-tions but rather by regulatory changes at the transcriptional level strongly suggests that MHC-I defects in human tumors are often reversible and can be corrected. Indeed, epigenetic modulation using DNA demethylating agents (e.g., 5-azacytidine and decitabine) and inhibitors of histone deacetylases (HDAC) (e.g., trichostatin A) restores MHC-I gene expression in tumor cells.2,13 Immunostimulatory cytokines, such as interferons, are also known to potently stimulate MHC-I-mediated

2012 Landes Bioscience.

250% when compared with untreated SKBR3 cells. Longer incubation periods of metformin treatment (i.e., 96 h) failed to further increase mitochondrial biogen-esis under the present experimental con-ditions. When an equivalent experiment was conducted in human BT-474 breast cancer cells, another well-characterized HER2 gene-amplified tumor model,39 metformin treatment was found to simi-larly enhance nDNA-encoded SDH-A and mtDNA-encoded COX-I expression by ~150% and 250%, respectively, com-pared with untreated BT-474 cells.

succinate-ubiquinone oxidoreductase. Interestingly, metformin treatment of HER2-overexpressing SKBR3 breast can-cer cells increased the expression of both COX-1 and SDH-A proteins in a dose-dependent manner (Fig. 1B ). Metformin concentrations as low as 100 μmol/L were sufficient to significantly upregulate mtDNA- and nDNA-encoded mitochon-drial markers in SKBR3 cells. Notably, while SDH-A expression was enhanced up to ~180% by treatment with 10 mmol/L metformin for 48 h, COX-1 expression was drastically upregulated by more than grow for 48 or 96 h in a metformin dilu-tion series. Next, given that mitochon-drial biogenesis involves the coordinated action of both nuclear- and mitochon-drial-encoded genomes, we measured the relative amounts of two representative subunits of different oxidative phosphory-lation enzyme complexes. Specifically, we examined the expression levels of cytochrome c oxidase I (COX-1) and mitochondrial succinate dehydrogenase (SDH-A), which are encoded by mito-chondrial DNA (mtDNA) and nuclear DNA (nDNA), respectively. COX-I, a subunit I of complex IV, is the component of the respiratory chain that catalyzes the reduction of oxygen to water; SDH-A is the 70-kDa subunit of complex II that serves as a major catalytic subunit of

Acknowledgements The work in the laboratory of Javier A.

Menendez is supported by the Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo, Fondo de Investigación Sanitaria (FIS), Spain, G rants CP05-00090 and PI06-0778 and RD06-0020-0028), the Fundación Científica de la Asociación Espa?ola Contra el Cáncer (AECC, Spain), and the Ministerio de Ciencia e Innovación (SAF2009-11579, Plan Nacional de I+D+I, MICINN, Spain). Alejandro Vazquez-Martin is the recipient of a Sara Borrell post-doctoral contract (CD08/00283, Ministerio de Sanidad y Consumo, Fondo de Investigación Sanitaria-FIS, Spain). Sílvia Cufí is the recipient of a Research Fellowship (Formación de Personal Investigador, FPI) from the Ministerio de Ciencia e Innovación (MICINN, Spain).References 1. Seliger B. Molecular mechanisms of MHC class

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After calculation of the MHC-I expres-sion index, as described above, we con-cluded that MFC-7/HER2 cells exhibited ~4.5-fold higher expression of cell sur-face-associated MHC-I when exposed to metformin; similarly, metformin-treated MCF10A/HER2 cells showed an approx-imately 1.5-fold increase in MHC-I expression over untreated cells.A causal association between oncogenic transformation, metabolic reprogram-ming and deficiencies in MHC-I antigen-restricted immunosurveillance suggests that intervention strategies aimed at reversing the bioenergetic signature of cancer cells might prevent their ability to evade the immune system. Here, we pro-vide the first evidence that metformin’s ability to enhance the tumor suppressor mechanism of mitochondrial biogen-esis 42-50 results in partial or complete res-toration of MHC-I expression in breast

cancer cells overexpressing the HER2

oncogene. Regardless of the underlying mechanism linking metformin-induced impairment of the reductive metabolic

program (mainly glycolytic) with restora-tion of impaired MHC-I surface expres-sion caused by oncogenic transformation,

additional studies should evaluate whether metformin treatment efficiently enhances

lysis rates of HER2-positive cancer cells by CTLs. Metformin’s ability to impede a pivotal strategy by which cancer cells

circumvent proper MHC-I expression may provide essential information for

the design of novel metformin-based immunotherapeutic strategies to com-bat HER2-overexpressing carcinomas.

Oncogene-mediated downregulation of components of the MHC-I antigen-pro-cessing pathway is associated with immune

escape of oncogene transformants in vivo and with disease progression. As such, we

suggest that our current findings delineate a previously unrecognized mechanism through which metformin (and met-formin-like drugs) may enable a cancer patient’s own immune system to mount an efficient anti-metastasis response that can prevent or delay disease recurrence. Finally, restored antigenicity and immu-nogenicity of tumor cells may represent a previously unrecognized primary mode of action underlying the undisputed cancer-preventive effects of metformin.

To determine whether metformin-enhanced mitochondrial biogenesis was associated with changes in the expres-sion status of MHC-I, the cell surface-associated levels of MHC-I were assessed by flow cytometry in SKBR3 cells stained with an anti-HLA-ABC monoclonal anti-body (Fig. 1C ). As a control, the cells were stained with the appropriate antibody iso-type. In untreated, HER2-overexpressing SKBR3 cells, MHC-I expression was almost undetectable by flow cytometry, whereas a marked increase of cell surface-associated MHC-I protein levels was observed in SKBR3 cells cultured with 1 mmol/L metformin. Next, the abundance of cell surface-associated MHC-I was quantitatively evaluated using an arbi-trary expression index obtained by multi-plying the MHC-I-associated Geo Mean (G M) fluorescence by the percentage (%) of MHC-I-positive cells (M1). After metformin treatment, MHC-I expres-sion drastically increased ~25-fold com-pared with the basal levels of MHC-I in untreated SKBR3 cells. These data are the first evidence showing that restoration of cell surface-associated MHC-I expression in previously MHC-I-negative, HER2-overexpressing tumor cells can be success-fully achieved solely by exposing cancer cells to the anti-diabetic biguanide met-formin. To further confirm metformin’s ability to upregulate MHC-I expression in different scenarios of HER2-driven breast cancer progression, we evalu-ated how metformin treatment altered MHC-I expression in non-tumorigenic, HER2-negative MCF-7 and MCF10A breast cancer epithelial cells engineered to overexpress the wild-type form of human HER2 oncogene via retroviral infection, a gene overexpression approach that avoids undesirable clone-related effects.40,41 We observed that treatment with 1 mmol/L metformin for 48 h significantly increased the MHC-I expression in these cells, albeit to a lesser extent than was observed in HER2 gene-amplified SKBR3 cells. We noticed that the cell surface expres-sion of MHC-I was similarly increased in MCF-7/HER2 and MCF10A/HER2 cells cultured in the presence of metfor-min, as revealed by a right shift in the expression curve when compared with the untreated control cells (Fig. 1C ).

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