JAK kinase inhibition abrogates STAT3 activation and head and neck squamous cell carcinoma tumor growth

Abstract

Aberrant activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) 3 has been implicated in cell proliferation and survival of many cancers including head and neck squamous cell carcinoma (HNSCC). AZD1480, an orally active pharmacologic inhibitor of JAK1/JAK2, has been tested in several cancer models. In the present study, the in vitro and in vivo effects of AZD1480 were evaluated in HNSCC preclinical models to test the potential use of JAK kinase inhibition for HNSCC therapy. AZD1480 treatment decreased HNSCC proliferation in HNSCC cell lines with half maximal effective concentration (EC50) values ranging from 0.9 to 4 μM in conjunction with reduction of pSTAT3(Tyr705) expression. In vivo antitumor efficacy of AZD1480 was demonstrated in patient-derived xenograft (PDX) models derived from two independent HNSCC tumors. Oral administration of AZD1480 reduced tumor growth in conjunction with decreased pSTAT3(Tyr705) expression that was observed in both PDX models. These findings suggest that the JAK1/2 inhibitors abrogate STAT3 signaling and may be effective in HNSCC treatment approaches.

Figure 1

(A) HNSCC cell lines express varying levels of pJAK1, pJAK2, and pSTAT3. HNSCC cell lines (OSC19, 686LN, HN5, UMSCC-1, UM-22B, PCI-15B, PCI-52, Cal33, and UM-22A) were seeded in 10-cm plates (1 × 10⁶ cells), and after 24 hours, cells were harvested to obtain cell lysates. Forty micrograms of protein/lane was subjected to electrophoresis and immunoblotted for pJAK1, pJAK2, pJAK3, and pSTAT3_Tyr705. β-Tubulin was used as a loading control. The numbers below the bands are a quantitative representation of pJAK1/β-tubulin, pJAK2/β-tubulin, pJAK3/β-tubulin, and pSTAT3_Tyr705/β-tubulin in HNSCC cell lines from three independent experiments. (B) Association between JAK2 mRNA expression and JAK2 copy number alteration in HNSCC tumors determined by GISTIC or RAE algorithm in the TCGA portal (*P < .0001; **P < .0001; ***P = .0168; ****P = .0069; *****P = .0307). P values were determined by unpaired t test with Welch’s correlation. (C) Association between JAK1 mRNA expression and JAK1 copy number alteration in HNSCC tumors determined by GISTIC or RAE algorithm in the TCGA portal (*P < .0001). P value was determined by unpaired t test with Welch’s correlation. (D) Association between JAK3 mRNA expression and JAK3 copy number alteration in HNSCC tumors determined by GISTIC or RAE algorithm in the TCGA portal (*P < .0001). P values were determined by unpaired t test with Welch’s correlation.

Figure 2

Proliferation of HNSCC cells upon knockdown of JAK2 by JAK2 siRNA. (A) UMSCC-1 and HN5 cells were transfected with JAK2 siRNA or control siRNA, and after 48 and 72 hours, cells were harvested for JAK2 expression. (B) Further cell proliferation was monitored upon JAK2 knockdown after days 1, 3, and 6. Results are from three separate experiments.

Figure 3

Effect of AZD1480 on pSTAT3_Tyr705 expression in HNSCC cell lines. UMSCC-1, HH5, Cal33, and UM-22A cells were treated with increasing concentrations of AZD1480. After 24 hours of treatment, cells were stimulated with IL-6 (50 ng/ml) for the last 15 minutes. At the end of the treatment, cells were harvested to obtain cell lysates. Forty micrograms of protein/lane was subjected to electrophoresis and immunoblotted for pSTAT3_Tyr705 and total STAT3. β-Tubulin was used as a loading control. The experiment was performed three times with similar results.

Figure 4

AZD1480 suppresses growth and expression of pSTAT3_Tyr705 target genes in HNSCC patient-derived heterotopic tumorgrafts. (A) HNSCC tumors from two individual patients were inoculated subcutaneously in the right and left flanks of NOD SCID gamma mice. Following the development of palpable tumors, mice were randomized and AZD1480 at a dose of 30 mg/kg was administered twice daily through oral gavage. Tumor volume measurements were obtained three times a week and measured to days 15 and 13 and are graphed. The nonparametric Wilcox Mann-Whitney test was used for tumor volume on day 15 to determine significance. (B) At the end of treatment (day 13 for patient 1 tumors and day 15 for patient 2 tumors), tumors were harvested, and whole-cell lysates were prepared and subjected to immunoblot analysis for pSTAT3_Tyr705 and STAT3. β-tubulin was used to assess protein loading. (C) The bar graph is a quantitative representation of the ratio of pSTAT3_Tyr705/β-tubulin (P = .003, patient 1 and P = .066, patient 2).

Figure S1.

Effect of AZD1480 on pSTAT3_Tyr705 expression in HNSCC cell lines. UMSCC-1 and Cal33 cells were treated with increasing concentrations of AZD1480. After 24 hours of treatment, cells were harvested to obtain cell lysates. Forty micrograms of protein/lane was subjected to electrophoresis and immunoblotted for pSTAT3_Tyr705 and total STAT3. β-Tubulin was used as a loading control. The experiment was performed three times with similar results.

Figure S2.

Effect of JAK2 siRNA on pSTAT3_Tyr705 expression in HNSCC cell lines. (A) UMSCC-1 and (B) HN5 cells were transfected with JAK2 siRNA or control siRNA, and after 48 hours and 72 hours, cells were harvested for pSTAT3_Tyr705 and STAT3 expression. β-Tubulin was used as a loading control. The experiment was performed three times with similar results.

Figure S3.

Effect of AZD1480 on pSTAT5_Tyr694 expression in HNSCC cell lines. Cal33 cells were treated with increasing concentrations of AZD1480. After 24 hours of treatment, cells were stimulated with IL-6 (50 ng/ml) for the last 15 minutes. At the end of the treatment, cells were harvested to obtain cell lysates. Forty micrograms of protein/lane was subjected to electrophoresis and immunoblotted for pSTAT3_Tyr694. β-Tubulin was used as a loading control. The experiment was performed three times with similar results.