Introduction
Hormonally Upregulated Neu-associated kinase is a member of the AMPK-family of protein kinases that was discovered in a RT-PCR-based screen to identify kinases expressed in the adult mammary gland [
1]. Previous findings indicate that HUNK is preferentially expressed in aggressive subsets of breast cancer and may play a pivotal role in mediating breast cancer metastasis [
2,
3]. Consistent with these assertions, our previous work shows that HUNK expression is regulated in response to HER2/neu activity and inhibiting this kinase impairs tumor formation and growth in primary HER2/neu tumor models [
4].
Although little is known about the intracellular function of HUNK, evidence is mounting that this kinase participates in the regulation of cell survival. Studies demonstrate that impairing HUNK in HER2/neu-positive (HER2+) and Akt-dependent mammary tumor models induces cell death by apoptosis [
4,
5]. Moreover, studies using
Hunk
−/−
(Hunk KO) mice show that normal mammary gland development is altered by loss of HUNK function during postlactational involution, a stage of mammary gland development governed by apoptotic clearance of mammary epithelial cells, where
Hunk KO mice display increased levels of apoptosis during involution [
5].
The process of autophagy has been linked to apoptosis [
6], and we have previously shown that HUNK mediates apoptosis [
4,
5]. However, a role for HUNK in autophagy has not been investigated. Because significant crosstalk exists between signaling pathways that regulate apoptosis and autophagy, in this study, we aimed to demonstrate that HUNK regulates autophagy in a manner consistent with its ability to regulate cell survival and show that the outcome of this activity impacts breast cancer resistance to HER2-targeted therapy.
Materials and methods
Cell culture
All cells were maintained at 37 °C and 5 % CO
2.
Hunk
+/+
(Hunk WT)and
Hunk
−/−
(Hunk KO) mammary gland fibroblasts (MGF) were isolated as previously described [
5] and were grown in DMEM (Hyclone) supplemented with 10 % super calf serum (SCS, Gemini). BT474 (ATCC) human breast cancer cells were grown in RPMI-1640 (Hyclone) supplemented with 10 % fetal bovine serum (FBS, Gibco). BT474 cells expressing control or HUNK shRNA (gift from Lewis Chodosh, University of Pennsylvania) were generated and maintained as previously described [
4]. JIMT-1 (Addex Bio) trastuzumab-resistant breast cancer cells were grown in DMEM (Hyclone) supplemented with 10 % FBS. JIMT-1 cells expressing control or HUNK shRNA were generated using the pGIPZ system (Thermo-GE/Dharmacon) and maintained in media containing 1 ug/ml puromycin. All media contained 2 mM glutamine (Thermo Scientific) and Penicillin/Streptomycin (Pen/Strep, Thermo Scientific) unless otherwise specified. pEGFP-LC3 was acquired through Addgene (plasmid #24920, provided by TorenFinkel [
7] ). Transfection of GFP-LC3 was performed using Turbofect (Thermo Scientific).
Immunoblotting
Cells were lysed in buffer containing final concentrations of 50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1 % Triton X-100; and 0.1 % SDS supplemented with HALT protease and phosphatase inhibitor cocktail (Thermo Scientific). For near-infrared imaging (Odyssey, LI-COR), secondary antibodies were purchased from Rockland Scientific. Primary antibodies used for western blotting are anti-LC3B (Cell Signaling- 2775), anti-HUNK [
4], and anti-
β-tubulin (Santa Cruz).
RNA isolation and quantitative Real-Time PCR
RNA was prepared using the GeneJet RNA isolation kit (Thermo Scientific). Reverse transcription was performed using the Maxima First Strand cDNA Synthesis Kit for RT-PCR (Thermo Scientific). The resulting cDNA was used to perform quantitative Real-Time PCR (QRT-PCR) using the Biorad myIQ system. Primers are HUNK-For-ATTAGCTTCCTGGAGGGGAC; HUNK-Rev-GTGATATTGGGGTGCGGAT and GAPDH-For-TGCACCACCAACTGCTTAGC; GAPDH-Rev-GGCATGGACTGTGGTCATGAG
Cell death analysis
Equal numbers of cells were plated and treated the following day by serum deprivation for 24 h or with 100 nM lapatinib (Santa Cruz) for 24 h prior to analysis by Caspase-3 activity assay (Sigma) or trypan blue exclusion and cell counting, which was performed using the cellometer mini cell counter (Nexcelom). For autophagy, cells were treated with 100 uM chloroquine (Sigma) for the indicated times and analyzed by Western blotting or microscopy (Nikon E800).
In vivo tumorigenesis
Animal care and all animal experiments were performed with the approval and in accordance with the guidelines of the Medical University of South Carolina IACUC. For orthotopic tumor analysis, 5 × 106 cells were injected in the abdominal mammary fat pat of immunocompromised mice (Nu/J-Foxn1nu/nu, Jackson Labs). Tumors were evaluated by manual palpation using calipers.
Statistical considerations for animal data
Tumor volume data from mouse experiments were analyzed using generalized estimating equations (GEEs) [
8,
9] with an identity link function and exchangeable correlation structure, a regression model that accommodates the lack of independence among measures obtained from the same mouse over time. Specifically, we modeled volume (mm
3) as a function of animal group (control versus HUNK shRNA), days from cell injection (measured as a continuous variable), and their two-way interaction. We used restricted cubic splines [
10] to transform days from cell injection due to observed non-linear trends in tumor volume over time. Comparisons between groups at each time point were performed using model-based linear contrasts. We used Kaplan–Meier survival curves to estimate group-specific median time to sacrifice, and compared survival times using a log-rank test. We used a similar approach to compare time to tumor volume of 100 mm
3. Analyses of animal data were performed using the R statistical software package [
11]. GEEs were fit using the geepack library in R [
12]. Kaplan–Meier curves and log-rank tests were conducted using the survival library in R [
13].
Discussion
Although a role for HUNK in promoting breast cancer initiation [
4,
5] and metastasis [
2,
3] has been uncovered, little is known about the intracellular function of this kinase. Evidence is mounting for a role for HUNK in regulating cell survival. Previous findings indicate that HUNK regulates apoptosis [
4,
5]. We now report that HUNK also regulates cell survival via autophagy. This finding is significant because autophagy is a mechanism by which breast cancer cells have been shown to survive treatment and acquire resistance [
14,
17,
20]. However, to some degree, the role of autophagy in human cancer is still unclear as this process is reported as both tumor promoting [
14,
17,
20‐
25] and tumor inhibitory [
26‐
28]. This paradox is exemplified in HER2+ breast cancer in which monoallelic deletion of the autophagy gene
BECN-
1 is frequently found yet these tumors are more sensitive to HER2-targeted therapy [
29]. This suggests that during early stages of tumor development, autophagy is tumor inhibitory but once a tumor is fully developed inhibiting autophagy makes treatment much more effective. Consequently, to clarify how to therapeutically target autophagy in breast cancer, carefully mapping the cell signaling pathways and molecules that control these processes is critical to develop effective breast cancer therapies.
Consistent with the idea that autophagy status influences tumor cell survival in response to treatment, autophagy competent tumor cells will adapt by utilizing autophagy to survive treatment, thus acquiring resistance. Therefore, being able to simultaneously induce tumor cell death by apoptosis and impair survival by autophagy has the potential to allow tumor cells to evade resistance. This strategy is directly applicable to HER2+ breast cancer because chronic application of HER2 inhibitors (i.e., trastuzumab and lapatinib) has been shown to lead to the up-regulation of autophagy response [
14,
17] and survival signaling pathways, like the PI3K/Akt pathway, in breast cancer cells to promote cell survival [
30]. Our new findings implicate HUNK as an attractive target because this kinase not only signals through HER2 [
4] and PI3K/Akt [
5] but we now show HUNK also regulates cell survival by impacting both apoptosis and autophagy.
While intriguing, additional investigation is warranted as we do not yet know the mechanism by which HUNK regulates autophagy and how this relates to apoptosis. It is possible that HUNK regulates these processes individually or alternatively and mediates crosstalk between the two pathways. We have previously demonstrated that HUNK can regulate p27 downstream of HER2 activation and attribute HUNK’s ability to promote survival, at least in part due to this function [
4]. Interestingly, p27 has been implicated in autophagy, and this function of p27 is directed by its phosphorylation at Threonine (T) 198 [
31]. The phosphorylation of p27 on T198 can be directed by multiple kinases including AMPK [
31], Akt [
32], and Rsk1 [
33]. AMPK-directed phosphorylation of p27 is associated with p27’s function toward autophagy [
31] whereas phosphorylation of p27 by Akt mediates cell survival [
32]. We have shown that HUNK is upregulated by Akt [
5]. Accordingly, it is possible that HUNK could have a selective function toward Akt-specific downstream signaling molecules, and it will be interesting to determine if HUNK regulates p27 in an Akt-dependent manner. Furthermore, it is perhaps appealing to speculate that an overlapping mechanism for HUNK exists toward Akt-specific substrates such as transcriptional regulation, modulation of protein localization, or sequestration of proteins in inactive complexes—perhaps by HUNK-directed phosphorylation. However, very little is known about the kinase activity of HUNK or its preferred phosphorylation consensus site. Consequently, this line of investigation is better suited for future studies.
In conclusion, our collective findings to date demonstrate that HUNK is a critical survival protein in HER2+ breast cancer cells. We have shown that HUNK regulates tumor cell survival by mediating apoptosis, and now we have also demonstrated that HUNK regulates survival by mediating autophagy. Taken together, these findings implicate HUNK as a potential target in HER2+ breast cancer that has the potential to be therapeutically beneficial in fighting resistance to HER2-targeted therapies.
Acknowledgments
The Yeh lab is supported by start-up funding from Medical University of South Carolina, by pilot research funding from an American Cancer Society Institutional Research Grant (IRG-97-219-14) from the American Cancer Society awarded to the Hollings Cancer Center, Medical University of South Carolina, by pilot research funding from a Department of Defense grant (W81XWH-11-2-0229) at the Medical University of South Carolina, and by an award from the Concern Foundation. These studies were also supported by the Biostatistics Shared Resource, Hollings Cancer Center, Medical University of South Carolina (P30 CA138313). ESY designed experiments, performed experiments, and wrote the paper. MAA performed experiments. EGH performed the statistical analysis of animal data and wrote the corresponding statistical methods section.
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