HUNK phosphorylates EGFR to regulate breast cancer metastasis

Epidermal growth factor receptor (EGFR) is commonly over-expressed in metastatic breast cancer yet metastatic breast cancer is generally resistant to anti-EGFR therapies, and the mechanism for resistance to EGFR inhibitors in this setting is not fully understood. Hormonally up-regulated neu-associated kinase (HUNK) kinase is up-regulated in aggressive breast cancers and is thought to play a role in breast cancer metastasis. However, no studies have been conducted to examine a relationship between EGFR and HUNK in breast cancer metastasis. We performed a kinase substrate screen and identified that EGFR is phosphorylated by HUNK. Our studies show that HUNK phosphorylates EGFR at T654, enhancing receptor stability and downstream signaling. We found that increased phosphorylation of T654 EGFR correlates with increased epithelial to mesenchymal, migration and invasion, and metastasis. In addition, we found that HUNK expression correlates with overall survival and distant metastasis free survival. This study shows that HUNK directly phosphorylates EGFR at T654 to promote metastasis and is the first study to show that the phosphorylation of this site in EGFR regulates metastasis

Correction: HUNK phosphorylates EGFR to regulate breast cancer metastasis

Erratum for 10.1038/s41388-019-1046-

Co-Targeting of JNK and HUNK in Resistant HER2-Positive Breast Cancer

<div><p>Strategies for successful primary treatment of HER2-positive breast cancer include use of the HER2 inhibitors trastuzumab or lapatinib in combination with standard chemotherapy. While successful, many patients develop resistance to these HER2 inhibitors indicating an unmet need. Consequently, current research efforts are geared toward understanding mechanisms of resistance and the signaling modalities that regulate these mechanisms. We have undertaken a study to examine whether signaling molecules downstream of epidermal growth factor receptor, which often act as compensatory signaling outlets to circumvent HER2 inhibition, can be co-targeted to overcome resistance. We identified JNK signaling as a potential area of intervention and now show that inhibiting JNK using the pan-JNK inhibitor, SP600125, is effective in the HER2-positive, resistant JIMT-1 xenograft mammary tumor model. We also investigate potential combination strategies to bolster the effects of JNK inhibition and find that co-targeting of JNK and the protein kinase HUNK can prohibit tumor growth of resistant HER2-positive mammary tumors <i>in vivo</i>.</p></div

HUNK knockdown inhibits JIMT-1 xenograft mammary tumor growth.

<p>A) Time to tumor size of 600 mm³ was significantly longer for mice with HUNKshRNA expressing tumors relative to control shRNA derived tumors based on Kaplan–meier survival curve estimates; p < 0.0001, log-rank test. B) Immunoblot analysis of control and HUNK shRNA expressing JIMT-1 cell lysates for phospho-JNK and total JNK expression. C) Immunoblot analysis of control and HUNK shRNA expressing JIMT-1 cell lysates for phospho-EGFR and total EGFR expression. D) Immunoblot analysis of control and HUNK shRNA expressing JIMT-1 cell lysates for phospho-AKT and total AKT expression E) Control and HUNK shRNA expressing JIMT-1 cells were treated with inhibitors targeting AKT, PLC, JNK, PI3K, MEK, and EGFR for 24 hrs and evaluated for Caspase-3 activity as a measure of apoptosis. p-values were determined by student’s T-test.</p

SP600125 inhibits mammary tumor growth of JIMT-1 xenograft tumors but combined lapatinib and JNK inhibition provides no added benefit.

<p>A) Immunoblot analysis of BT474 and JIMT-1 cell lysates for phospho-JNK and total JNK expression. B) Time to tumor size of 600 mm³ was significantly longer for SP600125 treated mice relative to placebo treated mice based on Kaplan–meier survival curve estimates; p = 0.002, log-rank test. n = 7, placebo. n = 8, SP600125. C) Immunoblot analysis of JIMT-1 cell lysates for phospho-JNK and total JNK after treatment of cells with DMSO, SP600125 (50 μM), lapatinib (1 μM), or a combination of SP600125 and lapatinib. D) JIMT-1 cells were treated with DMSO, SP600125 (50 μM), lapatinib (1 μM), or a combination of SP600125 and lapatinib for 24 hrs and evaluated for Caspase-3 activity as a measure of apoptosis. E) Time to tumor size of 600 mm³ was not different for mice treated with SP600125 (30 mg/kg), relative to mice treated with lapatinib (100 mg/kg) and SP600125 based on Kaplan–meier survival curve estimates. n = 10 animals per treatment group. p-value was not significant (n.s.) by log rank test.</p

Combined HUNK and JNK targeting induces cell death of JIMT-1 cells and impairs mammary tumor growth.

<p>A) Immunoblot analysis of control and HUNK shRNA expressing JIMT-1 cell lysates for LC3B I and II expression after SP600125 (50 μM), or SP600125 and chloroquine (100 ng/ μl) treatment to measure for autophagic flux. B) Quantitation of the fold change in LC3BII levels between the SP600125 and chloroquine treated cells and SP600125 alone treated cells in each cell type, control or HUNK shRNA, to determine the level of autophagic flux in each cell type. C) Time to tumor size of 600 mm³ was significantly longer for mice with HUNK shRNA derived tumors treated with SP600125 (30mg/kg) relative to mice with control shRNA derived tumors treated with SP600125 based on Kaplan–meier survival curve estimates; p <0.0001, log-rank test. n = 10, control. n = 10, HUNK shRNA.</p

SP600125 induces autophagy <i>in vitro</i> but choroquine does not have an additive effect on inhibiting mammary tumor growth when used with JNK inhibitor <i>in vivo</i>.

<p>A) Immunoblot analysis of JIMT-1 cell lysates for LC3B I and II after treatment of cells +/- chloroquine (left panel) as well as with DMSO, SP600125 (50 μM), or SP600125 and chloroquine (100 ng/ μl). (right panel) B) JIMT-1 cells were treated with DMSO, SP600125 (50 μM), chloroquine (100 ng/ μl), or a combination of SP600125 and chloroquine for 24 hrs and evaluated for Caspase-3 activity as a measure of apoptosis. C) Average tumor volume at day 14 after treatment of placebo treated versus chloroquine (50 mg/kg) treated animals harboring JIMT-1-derived xenograft mammary tumors. n = 10 animals per treatment group. p-values were determined by student’s T-test.</p

JNK inhibition with SP600125 in JIMT-1 cells induces cell death.

<p>A) BT474 cells or B) JIMT-1 cells treated with inhibitors targeting AKT, PLC, JNK, PI3K, SRC, mTORC1, p38, JAK, and c-RAF for 24 hrs and evaluated for Caspase-3 activity as a measure of apoptosis. p-values were determined by student’s T-test. n.s. = not significant</p