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Research Article

Metformin Prevents the Development of Oral Squamous Cell Carcinomas from Carcinogen-Induced Premalignant Lesions

Lynn Vitale-Cross, Alfredo A. Molinolo, Daniel Martin, Rania H. Younis, Takashi Maruyama, Vyomesh Patel, Wanjun Chen, Abraham Schneider and J. Silvio Gutkind
Lynn Vitale-Cross
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Alfredo A. Molinolo
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Daniel Martin
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Rania H. Younis
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Takashi Maruyama
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Vyomesh Patel
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Wanjun Chen
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Abraham Schneider
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J. Silvio Gutkind
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DOI: 10.1158/1940-6207.CAPR-11-0502 Published April 2012
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  • Figure 1.
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    Figure 1.

    Metformin induces HNSCC cell growth inhibition and reduces mTORC1 activity in an AMPK-independent manner. A, cell proliferation was assessed in HN12 cells treated with increasing doses of metformin or 100 nmol/L rapamycin (R) by [3H]-thymidine incorporation as described in Materials and Methods. ***, P < 0.01, vehicle- versus metformin-treated cells or rapamycin-treated cells. B, HN12 cell viability was assessed by the MTS colorimetric assay daily for 4 days. *, P < 0.05; **, P < 0.01 vehicle- versus metformin-treated cells. C and D, Western blot analysis to evaluate the dose- (C) and time- (D) dependent effects of metformin on the AMPK and mTORC1 pathways in HN12 cells, respectively. R, 100 nmol/L rapamycin as positive control for mTORC1 pathway inhibition. E, Western blotting to show the effects of the LKB1/AMPK signaling pathway on the response to metformin. Following transfections with a control siRNA (ctrl) or LKB1 siRNAs (1 and 2), 10 mmol/L metformin was added to HN12 cells for 24 hours. LKB1 RNAi was carried out as described in Materials and Methods. HeLa cells were used as positive controls to show lack of endogenous LKB1 activity. F, Western blotting to assess the LKB1-independent effect of the indicated doses of metformin on the AMPK and mTORC1 pathways in HeLa cells.

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    Figure 2.

    Optimization scheme of the 4NQO oral-specific chemical carcinogenesis model shows time-dependent malignant progression of oral epithelial dysplastic lesions. A, the indicated groups of mice (n = 20) were randomly divided and administered with 4NQO in the drinking water (50 μg/mL) for the indicated time (8, 10, 12, 14, and 16 weeks). Mice were then sacrificed or switched to regular water and sacrificed at week 22 from the initiation of the 4NQO administration. The tongue of each mouse was processed for detailed histologic examination as described in Materials and Methods. The average of the number of lesions per mouse, classified as low-grade dysplasia, high-grade dyplasia, or carcinoma based on histopathologic examination was recorded at the end of the 4NQO administration as well as on week 22 from the initiation of carcinogen exposure. B, representative photographs of lesions observed in mice at the indicated time since the initiation of carcinogen exposure in mice treated with 4NQO for 14 weeks. No evident gross alteration were observed at week 4; small elevated white lesions and discoloration are seen at week 8; the lesions grow to small berry-like elevations at week 16 and to ill-defined indurated white or congestive areas, often ulcerated, at week 22. Microscopically, the series of photograph depict the sequence of histologic changes on the dorsal tongue from a normal epithelium at week 4, through mild to moderate atypia at week 8, several dysplastic cellular alterations at week 16 (note the increasing thickness of the epithelium), to a fully infiltrating well-differentiated SCC at week 22.

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    Figure 3.

    Metformin decreases the number and size of oral tumors induced by orally administered 4NQO carcinogen. A, C57Bl/6 mice were administered 4NQO diluted in the drinking water for 14 weeks, as described in Materials and Methods, and remained on regular water until week 22. At week 14, the mice were divided into 2 groups and given either daily i.p. injections of metformin at a dose of 50 mg/kg/d or an equal volume of sterile saline for a period of 8 weeks. Grossly evident lesions started to appear by week 16, and by week 18 the difference between metformin-treated and -untreated groups was significant. The cumulative incidence of the number of macroscopic lesions per animal is shown; the different colors indicate their respective size: 2 to 4 mm (blue), 4 to 6 mm (orange), 6 to 8 mm (green), and 8 to 10 mm (red). B, gross morphology of the lesions developed in the animals treated with 4NQO in the metformin-treated and control untreated groups. At week 14, when 4NQO treatment was stopped, the alterations are more evident in the dorsal tongue. A rather extensive white and elevated tumor composed of 3 different outgrowths is evident in this control animal (arrow, left topmost photograph). Smaller lesions are seen in the ventral aspect of the tongue (arrow, bottom rightmost photograph). C, at week 22, an untreated control animal has developed a significant papillomatous tumor in the middle of the tongue as well as another similar lesion in the tip of the organ (arrows). Only small discolorations are observed in an animal treated with metformin. In the ventral tongue the control animal has developed 2 evident berry-like outgrowths, whereas only one small white papillomatous lesion is seen in the metformin-treated animal (arrows). D, at the conclusion of the experimental period (week 22), the difference in the incidence of tumor lesions per animal was evident between metformin- and control-treated mice and is significant.

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    Figure 4.

    Metformin decreases the number and size of oral tumors and prevents the malignant conversion of preneoplastic lesions. A, a sagittal section of a tongue in a control animal at week 22 after the initiation of the exposure to 4NQO in the drinking water for 14 weeks, where a total of 3 tumors are observed. The lesion closest to the tip of the tongue has a papillary (1) as well as a clearly infiltrative aspect (2); closer to the middle of the dorsal tongue another infiltrative SCC is evident (3). The third tumor, a papilloma, is observed in the ventral tongue (4). B, a higher magnification of lesion 2; atypical epithelial cells showing squamous differentiation (white arrowhead) infiltrate the skeletal muscle of the tongue (black arrowhead). They are surrounded by a dense inflammatory infiltrate (white star). C, in this metformin-treated animal a single large benign papillomatous lesion is seen in the posterior dorsal tongue (1). D, at a higher magnification, the squamous epithelium (white arrowhead) lines thin projections of the stroma (black arrowhead), it is evidently hyperplastic, although the cells display no evident atypia; mitotic figures are not evident in this photograph. E and F, tumor multiplicity, including benign and malignant tumors (E) as well as SCC multiplicity (F) are significantly different between metformin-treated and untreated control groups. G and F, the number of preneoplastic lesions per tongue, both low- and high-grade dysplasias was also significantly reduced in the metformin-treated animals.

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    Figure 5.

    Metformin decreases mTORC1 activity in the basal layer of oral epithelial dysplasias and mitotic activity in the hyperplastic epithelium. A, the percentage of proliferating cells was evaluated in hyperplastic tongue epithelium using a Ki-67 rat monoclonal antibody. The animals treated with metformin show a significantly lower level of proliferating cells as compared with the control. Representative images showing nuclear immunoreactivity for Ki-67 in metformin (left) and control-treated animals (right) as well as the quantification are shown in the figure. B, positive immunoreactivity to the mTORC1 downstream target pS6 in normal (normal) and hyperplastic (hyperplasia) tissues is limited to the upper layers of the squamous epithelium of the dorsal tongue (top). The rightmost photograph shows dysregulated expression in a SCC. Most of the cells express pS6, regardless of their degree of differentiation. Conversely, increased pS6 expression is clearly evident in premalignant dysplastic lesions developed in control animals treated for 14 weeks with 4NQO (dysplasia), where pS6 immunoreactivity appears on the basal and suprabasal epithelium (arrowheads), underscoring a decompartamentalization of mTORC1 activity within the proliferating epithelial layers (bottom left). One of the most interesting effects induced by metformin treatment is that it restores pS6 expression to the upper compartment of the squamous epithelium, far from the actively proliferating compartment (dysplasia, bottom right). Indeed, the arrows depict a marked increase in the number of pS6-positive cells in the lower epithelial compartment of dysplastic lesions in control animals in comparison with animals treated with metformin. C, these differences are more evident following an immunofluorescence double staining, in which pS6 expression (green fluorescence) is limited to the upper differentiated layers of the normal oral epithelium; in fact, no pS6 expression is seen in the basal and parabasal layers where K5 is highly expressed (red fluorescence; top left) in normal mice. In 4NQO-treated animals, the basal layers show a coexpression of both K5 and pS6 within the proliferating compartment as depicted by arrows pointing to yellow immunofluorescent cells (dysplasia, top right). Metformin significantly inhibits the expression of pS6 within the basal layers of mice exposed to 4NQO (dysplasia, bottom left). Moreover, there is a highly significant difference in the number of pS6-positive cells in the basal layer of the dysplastic lingual epithelium between metformin-treated and control animals exposed to 4NQO as shown in the bar graph (bottom right).

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  • Table 1.

    Effect of metformin administration on serum components and immune cell profiles

    ControlMetformin
    Blood (serum)n = 6n = 6
    Glucose, mg/dL103.8 ± 14.3107.3 ± 10.0
    Cholesterol, mg/dL99.8 ± 9.482.2 ± 9.0
    Triglycerides, mg/dL74.0 ± 9.280.3 ± 9.8
    Sodium, mmol/L153.3 ± 1.0152.5 ± 1.1
    Potassium, mmol/L6.9 ± 0.27.6 ± 0.5
    Chloride, mmol/L113.0 ± 2.1115.2 ± 0.6
    Creatinine, mg/dL0.1 ± 0.00.1 ± 0.0
    BUN, mg/dL24.2 ± 2.521.3 ± 0.9
    Calcium, mmol/L2.3 ± 0.02.3 ± 0.0
    Magnesium, mmol/L1.3 ± 0.01.2 ± 0.0
    Phosphorous, mg/dL7.9 ± 0.18.3 ± 0.3
    Alkaline phosphatase, U/L82.5 ± 6.288.2 ± 10.5
    ALT, U/L125.0 ± 29.7198.8 ± 56.7
    AST, U/L232.0 ± 20.1248.3 ± 14.3
    Amylase, U/L>650.0 ± 0.0>650 ± 0.0
    CK, total, U/L>800.0 ± 0.0>800 ± 0.0
    Lactate dehydrogenase, U/L>600.0 ± 0.0>600 ± 0.0
    Protein, total, g/dL5.3 ± 0.05.2 ± 0.2
    Uric acid, mg/dL3.5 ± 0.03.0 ± 0.1
    Spleen T-cell proliferationn = 8n = 8
    Media, 10−3 cpm3.9 ± 0.55.5 ± 1.5
    CD3, 10−3 cpm16.5 ± 1.627.8 ± 2.9b
    Spleen T cellsn = 8n = 8
    % total cells labeled
    CD4+22.9 ± 1.216.8 ± 1.5a
    CD8+29.8 ± 1.510.5 ± 1.9c
    TH112.6 ± 1.313.4 ± 1.1
    TH171.4 ± 0.13.4 ± 0.5b
    Tregs26.9 ± 1.127.2 ± 1.1

    NOTE: Mice were administered 4NQO diluted in the drinking water for 14 weeks, divided into 2 groups, and given either daily i.p. injections of metformin at a dose of 50 mg/kg/d or an equal volume of sterile saline for a period of 8 weeks, as described in Fig. 3. Mice were euthanized on week 22 from the initiation of 4NQO exposure, and serum and spleen T cells isolated as described in Materials and Methods. Serum components, spleen T-cell proliferation, and spleen T-cell subtypes were analyzed as described in Materials and Methods. Metformin exerted a very limited impact on serum components and metabolic markers and did not reduce the T-cell proliferative response. Metformin significantly enhanced TH17 cells without changing TH1 and Foxp3+ Tregs, an effect that is under current investigation.

    • ↵aP < 0.05.

    • ↵bP < 0.01.

    • ↵cP < 0.001.

Additional Files

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    Files in this Data Supplement:

    • Supplementary Figures 1-4 - PDF file - 92K
    • Supplementary Methods, Figure Legends 1-4 - PDF file - 910K
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Cancer Prevention Research: 5 (4)
April 2012
Volume 5, Issue 4
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Metformin Prevents the Development of Oral Squamous Cell Carcinomas from Carcinogen-Induced Premalignant Lesions
Lynn Vitale-Cross, Alfredo A. Molinolo, Daniel Martin, Rania H. Younis, Takashi Maruyama, Vyomesh Patel, Wanjun Chen, Abraham Schneider and J. Silvio Gutkind
Cancer Prev Res April 1 2012 (5) (4) 562-573; DOI: 10.1158/1940-6207.CAPR-11-0502

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Metformin Prevents the Development of Oral Squamous Cell Carcinomas from Carcinogen-Induced Premalignant Lesions
Lynn Vitale-Cross, Alfredo A. Molinolo, Daniel Martin, Rania H. Younis, Takashi Maruyama, Vyomesh Patel, Wanjun Chen, Abraham Schneider and J. Silvio Gutkind
Cancer Prev Res April 1 2012 (5) (4) 562-573; DOI: 10.1158/1940-6207.CAPR-11-0502
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