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. 2016 Apr 26;7(17):24228-41.
doi: 10.18632/oncotarget.8286.

mTOR inhibition as an adjuvant therapy in a metastatic model of HPV+ HNSCC

Joseph D Coppock  1 , Paola D Vermeer  1 , Daniel W Vermeer  1 , Kimberly M Lee  1 , W Keith Miskimins  1 , William C Spanos  1   2 , John H Lee  1   2
Affiliations

mTOR inhibition as an adjuvant therapy in a metastatic model of HPV+ HNSCC

Joseph D Coppock et al. Oncotarget. .

Abstract

Effective treatments for recurrent/metastatic human papillomavirus-positive (HPV+) head and neck squamous cell cancer (HNSCC) are limited. To aid treatment development, we characterized a novel murine model of recurrent/metastatic HPV+ HNSCC. Further analysis of the parental tumor cell line and its four recurrent/metastatic derivatives led to preclinical testing of an effective treatment option for this otherwise fatal disease. Reverse phase protein arrays identified key signaling cascades in the parental and recurrent/metastatic cell lines. While protein expression profiles differed among the recurrent/metastatic cell lines, activated proteins associated with the mTOR signaling cascade were a commonality. Based on these data, mTOR inhibition was evaluated as an adjuvant treatment for recurrent/metastatic disease. mTOR activity and treatment response were assessed in vitro by western blot, Seahorse, proliferation, clonogenic, and migration assays. Standard-of-care cisplatin/radiation therapy (CRT) versus CRT/rapamycin were compared in vivo. Low-dose rapamycin inhibited mTOR signaling, decreasing proliferation (43%) and migration (62%) while it enhanced CRT-induced cytotoxicity (3.3 fold) in clonogenic assays. Furthermore, rapamycin re-sensitized CRT-resistant, metastatic tumors to treatment in vivo, improving long-term cures (0-30% improved to 78-100%, depending on the recurrent/metastatic cell line) and limiting lymph node metastasis (32%) and lung metastatic burden (30 fold). Studies using immune compromised mice suggested rapamycin's effect on metastasis is independent of the adaptive immune response. These data suggest a role of mTOR activation in HPV+ HNSCC recurrent/metastatic disease and that adjuvant mTOR inhibition may enhance treatment of resistant, metastatic cell populations at the primary site and limit distant metastasis.

Keywords: head and neck oral cancer; human papillomavirus; mTOR; metastasis; rapamycin.

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Conflict of interest statement

The authors report no financial or other conflicts of interest relevant to the subject of this article.

Figures

Figure 1
Figure 1. mTOR signaling in HPV+ OPSCC recurrent/metastatic disease
RPPA results highlighting that mTOR activation is present in parental mEERL and all recurrent/metastatic MLM cell lines, as indicated by the fact that clusters of the highest levels of proteins (red) are activated, phosphorylated mTOR signaling proteins and downstream targets. Importantly, mTOR signaling is inhibited in all lines by rapamycin alone and in combination with CRT (shift to green). The full array for mEERL is shown as an example. The arrow indicates the region enlarged for each cell line individually below, showing changes under the indicated treatment conditions. Full-length arrays for each cell line can be found in Supplementary Figure 1.
Figure 2
Figure 2. mTOR and metabolic activity are comparable or elevated in recurrent/metastatic HPV+ OPSCC cell lines
(A) Western blot analysis of the mTOR pathway in each of the MLM cell lines and parental mEERL with and without rapamycin treatment. (B) Comparison of metabolic levels of each MLM cell line compared to parental mEERL, measured as ECAR by Seahorse. All MLM cell lines have metabolic levels comparable to mEERL (MLM3 and MLM10 ns) or elevated (MLM1 and MLM5 *p < 0.04). (C) Cell proliferation assays showing a dose response to rapamycin treatment in all lines and significant inhibition of cell growth even at low-dose (10 nM) rapamycin (*p ≤ 0.05 to control; **p ≤ 0.05 to control and **p ≤ 0.021 to 10 nM). SD is shown by error bars.
Figure 3
Figure 3. Rapamycin enhances effects of cisplatin & radiation, sensitizing recurrent/metastatic cells to treatment
(A) Representative images of colonies of mEERL and each MLM cell line formed with no treatment, CRT, rapamycin, or their combination in clonogenic assays. Representative images for MLM1 and MLM10 can be found enlarged to show detail in Supplementary Figure 2. (B) Average colony number (Top; *p < 0.044 to control, †p < 0.03 to control, ‡p < 0.02 to control, #p < 0.02 to individual treatments, **p < 0.02 to CRT only) and size (Bottom; *p < 0.006 to control, †p < 0.03 to control, ‡p < 0.003 to control, #p < 0.05 to individual treatments, **p < 0.05 to CRT only) from the triplicate wells represented in A as a fraction of control. (C) Average number (Top; *p < 0.047 to control, †p < 0.044 to control, ‡p < 0.008 to control, #p ≤ 0.05 to individual treatments) and size (Bottom; *p < 0.003 to control, †p < 0.03 to control, ‡p < 0.0009 to control, #p ≤ 0.05 to individual treatments) of colonies of mEERL and each MLM cell line formed in clonogenic assays as above but instead using cisplatin alone, without radiation. (D) Crystal violet assays using two HPV+ (SCC47 & SCC90) and two HPV- (SCC1 and SCC84) human HNSCC cell lines. Absorbance of crystal violet destain solution, a surrogate of total cell number, is shown as a fraction of control for each cell line under each of the indicated treatment conditions (*p < 0.006 to control, ‡p < 0.04 to individual treatments, †p < 0.04 to cisplatin only, #p < 0.02 to cisplatin/rapamycin). SD is shown by error bars.
Figure 4
Figure 4. Rapamycin re-sensitizes recurrent/metastatic cell lines to treatment in vivo, significantly prolonging survival and improving long-term cures
(A) Average tumor volume per cell line and treatment group. Solid lines represent tumor growth of CRT-treated mice, and dotted lines CRT/rapamycin treated mice. Both CRT and CRT/rapamycin treated mEERL tumors responded completely to treatment (ns to each other), while CRT-treated MLM tumors all showed significant resistance compared to mEERL (p < 0.021). The addition of rapamycin significantly re-sensitized all MLM tumors to treatment, evident in the separation of CRT to CRT/rapamycin treated MLM tumor growth curves (p < 0.001). P-values represent comparisons of average endpoint tumor volumes. N = 10 mice per group. (B) Survival plots for each tumor type comparing CRT to CRT/rapamycin treated mice. All CRT-treated mice with MLM tumors had significantly worse survival compared to mEERL tumor-bearing mice (p ≤ 0.04), indicating treatment resistance. However, the addition of rapamycin significantly improved survival with all MLM tumor types (p < 0.021; mEERL CRT to CRT/rapamycin ns).
Figure 5
Figure 5. Adjuvant rapamycin limits lymph node and lung metastasis
(A) Representative images of pan-cytokeratin staining (by IHC) of metastases in fixed, paraffin embedded lung tissue sections from each mouse treated with CRT (−Rapa) or CRT/rapamycin (+Rapa). Images were taken at 4x magnification, with insets at 40x to show staining. Arrows indicate metastases. (B) Correlating average number of lung metastases per mouse, averaged for each tumor type and treatment group. All MLM tumors gave rise to a significantly increased average number of lung metastases compared to parental mEERL with CRT alone (p < 0.03). CRT plus adjuvant rapamycin significantly reduced the number of lung metastases in all MLM groups (p < 0.02). SEM is shown by error bars. (C) Cytokeratin IHC showing an example of a positive draining inguinal lymph node of each MLM tumor type alongside a representative node from a mouse with a mEERL tumor. Images are at 4×, with insets at 40× to show staining. (D) Percent of lung metastasis positive mice and lymph nodes from each tumor type and treatment group.
Figure 6
Figure 6. Metastasis and the ability of rapamycin to limit it are independent of the adaptive immune response
(A) Average tumor growth and (B) survival data from MLM3 tumors treated with CRT and CRT/rapamycin in immunocompromised (Rag) mice. N = 10 mice per group. Nearly identical responses to those observed in wild-type mice resulted. Neither tumor growth nor survival were significantly different from wild-type mice under either treatment condition (Figure 4), and the addition of rapamycin still led to significantly inhibited tumor growth (p < 3.59 × 10−5) and improved survival (p < 0.001). Regarding tumor growth, p-values represent comparisons of average endpoint tumor volumes. (C) Representative images of correlating cytokeratin stained metastases in lung tissue alongside quantifications indicating a significantly reduced metastatic burden at the lungs and decreased number of metastasis positive mice with adjuvant rapamycin, as in wild-type mice. Images were taken at 4x magnification, with insets at 40x to show staining. Arrows indicate metastases. (D) In vitro migration assay as a basic measure of metastatic potential in the indicated cells treated +/− low-dose (10 nM) rapamycin. Rapamycin significantly limited migration of the metastatic MLM cells (*p < 0.05), but did not affect the migration of mEERL, which were very minimally migratory under either condition. SD is shown by error bars.
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