Sustained Src inhibition results in signal transducer and activator of transcription 3 (STAT3) activation and cancer cell survival via altered Janus-activated kinase-STAT3 binding

Abstract

Locoregional and distant recurrence remains common and usually fatal for patients with advanced head and neck squamous cell carcinoma (HNSCC). One promising molecular target in HNSCC is the Src family kinases (SFK). SFKs can affect cellular proliferation and survival by activating the signal transducer and activator of transcription (STAT) family of transcription factors, especially STAT3. Surprisingly, sustained SFK inhibition resulted in only transient inhibition of STAT3. We investigated the mechanism underlying STAT3 activation and its biological importance. Specific c-Src knockdown with small interfering RNA (siRNA) resulted in STAT3 activation showing specificity, which was inhibited by Janus-activated kinase (JAK; TYK2 and JAK2) depletion with siRNA. Sustained SFK inhibition also resulted in recovered JAK-STAT3 binding and JAK kinase activity after an initial reduction, although JAK phosphorylation paradoxically decreased. To determine the biological significance of STAT3 activation, we combined specific STAT3 depletion with a pharmacologic SFK inhibitor and observed increased cell cycle arrest and apoptosis. Likewise, the addition of STAT3- or JAK-specific siRNA to c-Src-depleted cells enhanced cytotoxicity relative to cells incubated with c-Src siRNA alone. These results show that reactivation of STAT3 after sustained, specific c-Src inhibition is mediated through altered JAK-STAT3 binding and JAK kinase activity and that this compensatory pathway allows for cancer cell survival and proliferation despite durable c-Src inhibition. To our knowledge, this novel feedback pathway has never been described previously. Given that pharmacologic SFK inhibitors are currently being evaluated in clinical trials, these results have potential clinical implications for cancer therapy.

Figure 1

SFK inhibition results in transient STAT3 inhibition followed by STAT3 reactivation in vitro and in vivo. (A) Six HNSCC cell lines were incubated with 100 nM dasatinib for the indicated times and analyzed by Western blotting. (B) Western blots for activated STAT3 (pSTAT3, Y705) were analyzed using densitometry and normalized to β-actin. Bars represent standard deviation. *p ≤ 0.05 vs. control. (C) Cells were incubated with 100 nM dasatinib for the indicated times and analyzed by Western blotting with pSTAT3 (serine 727). (D) Mice bearing human HNSCC orthotopic xenografts were treated with dasatinib and euthanized at the the indicated times. Tumors were lysed and subjected to Western blotting.

Figure 2

SFK inhibition or depletion results in STAT3 reactivation in vitro. (A) STAT3 DNA binding was measured in nuclear extracts from cells incubated with 100 nM dasatinib. (B) STAT3 transcription was measured using a luciferase reporter assay in Tu167 cells incubated with 100 nM dasatinib. (C) Tu167 cells were transfected with c-Src–specific siRNA or controls and then analyzed by Western blotting. (D) Western blots from Fig. 2C were analyzed using densitometry and normalized to β-actin expression. Bars represent standard deviation. *p ≤ 0.05 vs. control.

Figure 3

The effects of JAK family member knockdown on STAT3 activation. (A, B) Tu167 cells were transfected with scrambled or specific siRNA as indicated and analyzed by Western blotting. Dasatinib or vehicle control was added for 7 h in the indicated samples. (C) Western blots for activated STAT3 (Y705) were analyzed using densitometry.

Figure 4

The effects of c-Src inhibition on JAK phosphorylation, kinase activity, and STAT3 binding. (A) Tu167 cells were incubated with 100 nM dasatinib and analyzed by Western blotting. (B) Tu167 cells were transfected with c-Src–specific siRNA or controls and then analyzed by Western blotting. (C) JAK2 was immunoprecipitated from Tu167 cells treated with the indicated agents and subjected to an in vitro kinase assay. (D) Tu167 cells were incubated with 100 nM dasatinib or 2.5 μM pyridone 6 and immunoprecipitated with either the TYK2 or JAK2 antibodies, as indicated. The immunoblots were then probed with the indicated antibodies.

Figure 5

STAT3 depletion enhances the biologic effects of SFK inhibition or depletion. (A) Tu167 cells were transfected with STAT3-specific siRNA or controls and analyzed by Western blotting. (B, C) Cells were incubated with 40 (Tu167) or 200 (Tu686) nM dasatinib for 24 h starting 48 h after transfection with STAT3 siRNA or controls. For the cell cycle analysis (B), cells were stained with propidium iodide and analyzed by FACS; the proportion of cells in the S phase is graphed as a percentage of the total. For the apoptosis assay (C), cells were analyzed by TUNEL staining. Bars represent standard deviation. *p ≤ 0.05 vs. vehicle-treated control; **p ≤ 0.05 vs. dasatinib-treated cells with control siRNA).

Figure 6

Depletion of STAT3 enhances cytotoxicity and the downstream signaling effects of c-Src depletion. (A) Tu167 cells were transfected with STAT3-specific siRNA, c-Src–specific siRNA, both, or controls. The number of viable cells was estimated 96 h after transfection with an MTT assay and expressed as the amount of change from control levels (vehicle alone). (B) Tu167 cells were transfected with TYK2- and JAK2-specific siRNA, c-Src–specific siRNA, all three, or controls. The number of viable cells was estimated 96 h after transfection with an MTT assay and expressed as the amount of change from control levels (vehicle alone). Bars represent standard deviations. Tu167 (C) or Tu686 (D) cells that were transfected with STAT3 siRNA or controls were incubated with 40 (C) or 200 (D) nM dasatinib for 24 h then lysed and analyzed by Western blotting.