Article Figures & Data
Figures
- Figure 1.
A, HNSCC cell lines CAL27, CAL33, and UMSCC47 were treated with the indicated concentrations of metformin or rapamycin, lysed and analyzed by Western blotting for expression of pS6/S6, pAMPK/AMPK, pAKT/AKT, and α-tubulin as a loading control. B, cells were treated as described in A) in triplicate for each condition. pS6 levels were analyzed by Western blotting, quantified and normalized to S6. Values are the ratio of the means to the control ± SEM. C, [3H]-thymidine incorporation assay of CAL27, CAL33, and UMSCC47 cells treated with metformin or rapamycin. D, colony formation assay of the HNSCC cell lines treated with metformin or rapamycin. **, P < 0.01.
- Figure 2.
A, schematic representation of time scale for the metformin treatment of mouse HNSCC xenograft models. B, metformin was delivered to mice through drinking water at the indicated concentrations for 4 days. Plasma from mouse blood was isolated, and metformin plasma concentration was measured. Sem-transparent gray area indicates typical range of metformin concentrations in plasma of diabetic patients and healthy control individuals treated with metformin. C, injected cells are indicated at the top left corner of each panel. Left, time course of tumor growth with tumor volumes measured over time. Control tumor growth measurements are shown in green circles, and tumor growth measurements on metformin-treated mice are shown in red squares. Middle, average weight of tumors at the endpoint of the experiment. Right, H&E staining of tumor sections with control tumors shown on top and the tumors from metformin-treated mice shown at the bottom. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
- Figure 3.
UMSCC47 cells were injected into immunedeficient mice as described in Fig. 2. When the mice developed palpable tumors, they were divided into three groups (n = 6 tumors for each group). The control group did not receive any treatment, while other groups were treated for three days with metformin in the drinking water (2.5 mg/mL) or with rapamycin at 5 mg/kg/d by intraperitoneal (i.p.) injections. A, Western blotting analysis of changes in mTOR signaling pathway, showed here in two separate tumors per treatment group. B and C, quantification of percentage of pS6+ (B) and BrdUrd+ (C) cells from the stained sections shown in D. D, tumor stainings with H&E and IHC for pS6 and BrdUrd.
- Figure 4.
UMSCC47 cells were infected with lentiviruses expressing a pool of shRNAs targeting human organic cation transporter (OCT3/SLC22A3) or nonspecific control shRNAs. The UMSCC47-derived control and OCT3 shRNA cells were analyzed side by side for metformin effects on the cells. A, Western blotting analysis of OCT3 expression in the HNSCC cell lines (left) and UMSCC47-derived control and OCT3 shRNA cells (right). B, top, UMSCC47-derived cells were treated with metformin or rapamycin as described in Fig. 1A, and expression of pS6/S6, pAMPK/AMPK was analyzed by Western blotting. Metformin was used at 1 mmol/L concentration, α-tubulin was used as a loading control. Bottom, mice bearing UMSCC47-derived control or OCT3 shRNA tumors were treated for 3 days with metformin or rapamycin as described in Fig. 3. The tumors were isolated and analyzed by Western blotting for pS6, S6, pAMPK, AMPK, and α-tubulin expression. C, quantification of pS6 expression levels normalized to S6 with level in control set as 100% (n = 3). D, [3H]-thymidine incorporation experiment was performed as described in Fig. 1C with metformin at 1 mmol/L concentration. The data were normalized to control values set as 100% (n = 4). E, colony formation assay was performed as described in Fig. 1D with metformin at 1 mmol/L concentration. The data were normalized to control values set as 100% (n = 4). F, time course of tumor growth with tumor volumes measured over time. Control UMSCC47 tumors shown in continuous lines, UMSCC47-OCT3 shRNA tumors shown in dotted lines. Untreated tumors are depicted as green lines and metformin-treated tumors are depicted as red lines. G, expression of OCT3 in a panel of tumors described in B. Three independent tumors derived from UMSCC47 control shRNA and OCT3 shRNA were used for the Western blot analysis, using α-tubulin as a loading control. H, average weight of tumors at the endpoint of the experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
- Figure 5.
A panel of human HNSCC patient samples was analyzed for expression of pS6 and OCT3 as biomarkers indicating mTOR signaling activation and potential sensitivity to metformin, respectively. A, IHC staining of normal epithelium and HPV− and HPV+ HNSCC tumor sections for pS6, p16, and OCT3. B–D, quantification of the IHC staining results shown in A. B, the percentage of pS6+ cells in normal tissues and in p16− and p16+ HNSCC tumors. C, the percentage of p16+ cases in normal tissues, HPV− and HPV+ HNSCC tumors. D, the percentage of OCT3+ cases in normal tissues and HPV− or HPV+ HNSCC tumors. The number of tissues analyzed is indicated in each case. ***, P < 0.001; ns, not significant.
- Figure 6.
A series of oral and cervical cancer TMAs including histologic cores from lesions arising in HIV+ patients were evaluated for p16 (HPV surrogate marker), pS6, and OCT3 expression by IHC. In these studies, we also included representative primary cases of HPV+ oral, uterine cervix, and perianal SCC lesions from HIV+ patients. A, IHC staining of HIV+ oral SCC showing p16, pS6, and OCT3 IHC stainings. Staining of a representative HPV+ sample is shown on the left, and HPV− on the right. B, quantification of the percentage of p16+ cases in HIV+ and HIV− HNSCC tissues. C, quantification of the percentage of pS6+ cells in HIV+ and HIV− HNSCC tissues. D, quantification of the percentage of OCT3+ cells in HIV+ and HIV− HNSCC tissues. The number of tissues evaluated in each case is indicated. **, P < 0.01; NS, no statistically significant difference. E, representative staining of cervical and anal SCC with H&E and IHC for p16, pS6, and OCT3. **, P < 0.01; ns, not significant.
Additional Files
Supplementary Data
- Supplemental Figure 1 - Figure S1. HNSCC cell lines CAL27, CAL33 and UMSCC47 were treated with the indicated concentrations of metformin or with rapamycin for colony formation assay (see Materials and Methods).
- Supplemental Figure 2 - Figure S2. A representative anal tumor section showing H&E staining and pS6, Ki67, p16 and OCT3 expression by IHC.
- Supplemental Materials - Supplemental Materials and Methods: Reagents, cell lines, tissue culture. Legends to figure S1 and S2.
Volume 8, Issue 3
