Small-molecule targeting of signal transducer and activator of transcription (STAT) 3 to treat non-small cell lung cancer

Abstract

Objective: Lung cancer is the leading cause of cancer death in both men and women. Non-small cell lung cancer (NSCLC) has an overall 5-year survival rate of 15%. While aberrant STAT3 activation has previously been observed in NSCLC, the scope of its contribution is uncertain and agents that target STAT3 for treatment are not available clinically.

Methods: We determined levels of activated STAT3 (STAT3 phosphorylated on Y705, pSTAT3) and the two major isoforms of STAT3 (α and β) in protein extracts of 8 NSCLC cell lines, as well as the effects of targeting STAT3 in vitro and in vivo in NSCLC cells using short hairpin (sh) RNA and two novel small-molecule STAT3 inhibitors, C188-9 and piperlongumine (PL).

Results: Levels of pSTAT3, STAT3α, and STATβ were increased in 7 of 8 NSCLC cell lines. Of note, levels of pSTAT3 were tightly correlated with levels of STAT3β, but not STAT3α. Targeting of STAT3 in A549 cells using shRNA decreased tSTAT3 by 75%; this was accompanied by a 47-78% reduction in anchorage-dependent and anchorage-independent growth and a 28-45% reduction in mRNA levels for anti-apoptotic STAT3 gene targets. C188-9 and PL (@30 μM) each reduced pSTAT3 levels in all NSCLC cell lines tested by ≥50%, reduced anti-apoptotic protein mRNA levels by 25-60%, and reduced both anchorage-dependent and anchorage-independent growth of NSCLC cell lines with IC50 values ranging from 3.06 to 52.44 μM and 0.86 to 11.66 μM, respectively. Treatment of nude mice bearing A549 tumor xenografts with C188-9 or PL blocked tumor growth and reduced levels of pSTAT3 and mRNA encoding anti-apoptotic proteins.

Conclusion: STAT3 is essential for growth of NSCLC cell lines and tumors and its targeting using C188-9 or PL may be a useful strategy for treatment.

Keywords: NSCLC; STAT3; Small molecule inhibitors.

Figure 1. STAT3 is constitutively activated in most NSCLC cell lines, which correlates with levels of STAT3β

A) Level of pSTAT3 were determined by Luminex beads in human NSCLC cell lines and in a normal human bronchial epithelial cell line (HBEC3-KT); values were corrected for GAPDH levels, expressed as a fraction of that obtained in HBEC3-KT cells, and the mean ± SD of 3 determinations shown; an asterisk (*) indicates those cell lines increased compared to HBEC3-KT cells (p<0.04). B) Levels of STAT3α, STAT3β and GAPDH were determined by immunoblot in lysates of murine embryonic fibroblast cells (MEF) lines in which the STAT3 gene was deleted followed by transient expression of STAT3α (MEF-STAT3α) or STAT3β (MEF-STAT3β)(13), HBEC3-KT cells, and human NSCLC cell lines (representative gel shown). Levels of STAT3α (C) and STAT3β (D) were quantified by densitometry using Image software. Each value was corrected using its corresponding GAPDH level and the mean ± SD of 2 determinations shown for each cell line (left panels) or as a function of its corresponding pSTAT3 level (right panels). An asterisk (*) indicates those cell lines increased compared to HBEC3-KT cells (p<0.02). pSTAT3 levels did not significantly correlate with levels of STAT3α (R²=0.1856, p=0.247), but did with levels STAT3β levels (R²=0.7348, p=0.0031).

Figure 2. Effect of shRNA targeting of STAT3 on STAT3 gene targets and A549 cell proliferation

Total RNA (A) or protein (B) was extracted from A549 cells stably transfected with either control shRNA or 2 distinct STAT3 shRNA constructs and used to determine levels of STAT3 mRNA by quantitative RT-PCR (A) or levels of pSTAT3, total STAT3 and GAPDH protein using Luminex beads (B); mean ± SD of 3 separate determinations are shown; *, p<0.003 for each. C) Cell proliferation in A549 cells stably transfected with either control shRNA or 2 distinct STAT3 shRNA constructs was determined by MTT assay using either anchorage-dependent (left panel) and anchorage- independent (right panel) conditions. Data shown are mean ± SD of 3 determinations; *, p<0.0001 for each. D) Levels of four STAT3 downstream gene targets were determined in total RNA of A549 cells stably transfected with either control shRNA or 2 distinct STAT3 shRNA constructs using quantitative RT-PCR. Data shown are mean ± SD of 3 determinations; *, p<0.02, p<0.04, p<0.03 and p<0.02, as shown.

Figure 3. C188-9 or PL treatment decreased levels of pSTAT3, reduced proliferation, and induced apoptosis of A549 cells

A549 cells were incubated with C188-9 (left panel) or PL (right panel; @30μM) for the indicated time periods (A) or for 2.5 hrs at the indicated concentrations (B). pSTAT3 levels were determined in duplicate using Luminex beads, corrected for GAPDH. Data presented are mean ± SD of duplicate determinations; * indicates treatment time intervals (A) and concentrations (B) significantly reduced from time 0 or concentration=0 (p<0.04). C) A549 cells were incubated in C188-9 (left panel) or PL (right panel) at the concentrations indicated for 72 hrs under anchorage-dependent (top panel) and anchorage-independent (bottom panel) conditions. Cell proliferation was assayed by MTT assay and the results used to generated the curves and IC₅₀ values shown. D) Levels of four STAT3 downstream gene targets were determined in total RNA of A549 cells treated for 4 hours with C188-9 or PL (@30μM) using quantitative RT-PCR. Data shown are mean ± SD of 3 determinations; an asterisk (*) indicates a significant difference from untreated cells with p values indicated.

Figure 4. Annexin V staining of A549 cells following incubation with C188-9 or PL

A549 cells were incubated with C188-9 and PL (@30μM) for 24 hours then stained with Annexin V and analyzed by flow cytometry. The percentage of cells in the necrotic, live, and apoptotic-gated areas are indicated.

Figure 5. Effect of C188-9 and PL on A549 xenograft tumor volume, tumor weight, pSTAT3 levels, and expression of downstream STAT3 gene targets

Tumor volumes (A) or weights (B) were measured on the days indicated or following treatment for 3 weeks, respectively, with C188-9 (50mg/kg IP twice daily; n=12), PL (30mg/kg IP daily; n=11), or vehicle (n=17). The mean ± SD for each group is shown. In panel A, asterisk (*) or caret (^) indicates a reduction compared to vehicle in animals treated with C188-9 (p<0.04) or with PL (p<0.04), respectively. In panel B, the asterisk (*) indicates p<0.05. C) Levels of pSTAT3 and GAPDH levels were determined using Luminex beads in protein extracts of tumors; pSTAT3 levels were normalized for GAPDH and the mean ± SD shown; the asterisk (*) indicates a reduction in pSTAT3 levels in C188-9- or PL-treated tumors compared to vehicle (p<0.039). D) Levels of STAT3 downstream genes targets were assessed by RT-PCR in total tumor RNA. Mean ± SD is shown; an asterisk (*) indicates a significant reduction in Bcl-2 (p<0.05), Bcl-xL (p<0.045), Cyclin D1 (p<0.022) and Survivin (p<0.02) RNA levels in C188-9- and PL-treated tumors compared to vehicle-treated tumors.

Figure 6. Effect of C188-9 or PL treatment on levels of pSTAT3 levels in PBMC

Levels of pSTAT3 and total STAT3 were determined using Luminex beads in protein extracts of PBMC isolated from whole blood of tumor-bearing mice after 3 weeks of treatment with vehicle, C188-9, or PL. Level of pSTAT3 were normalized to total STAT3. The mean ± SD is shown; an asterisk (*) indicates a reduction in pSTAT3 level compared to vehicle (p=0.039).