Core-Fucosylated Tetra-Antennary N-Glycan Containing A Single N-Acetyllactosamine Branch Is Associated with Poor Survival Outcome in Breast Cancer.

(1) Glycoproteins account for ~80% of proteins located at the cell surface and in the extracellular matrix. A growing body of evidence indicates that α-L-fucose protein modifications contribute to breast cancer progression and metastatic disease. (2) Using a combination of techniques, including matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) based in cell and on tissue imaging and glycan sequencing using exoglycosidase analysis coupled to hydrophilic interaction ultra-high performance liquid chromatography (HILIC UPLC), we establish that a core-fucosylated tetra-antennary glycan containing a single N-acetyllactosamine (F(6)A4G4Lac1) is associated with poor clinical outcomes in breast cancer, including lymph node metastasis, recurrent disease, and reduced survival. (3) This study is the first to identify a single N-glycan, F(6)A4G4Lac1, as having a correlation with poor clinical outcomes in breast cancer.

HUNK as a key regulator of tumor-associated macrophages in triple negative breast cancer.

Triple-negative breast cancer (TNBC) lacks the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). TNBC tumors are not sensitive to endocrine therapy, and standardized TNBC treatment regimens are lacking. TNBC is a more immunogenic subtype of breast cancer, making it more responsive to immunotherapy intervention. Tumor-associated macrophages (TAMs) constitute one of the most abundant immune cell populations in TNBC tumors and contribute to cancer metastasis. This study examines the role of the protein kinase HUNK in tumor immunity. Gene expression analysis using NanoString's nCounter PanCancer Immune Profiling panel identified that targeting HUNK is associated with changes in the IL-4/IL-4 R cytokine signaling pathway. Experimental analysis shows that HUNK kinase activity regulates IL-4 production in mammary tumor cells, and this regulation is dependent on STAT3. In addition, HUNK-dependent regulation of IL-4 secreted from tumor cells induces polarization of macrophages into an M2-like phenotype associated with TAMs. In return, IL-4 induces cancer metastasis and macrophages to produce epidermal growth factor. These findings delineate a paracrine signaling exchange between tumor cells and TAMs regulated by HUNK and dependent on IL-4/IL-4 R. This highlights the potential of HUNK as a target for reducing TNBC metastasis through modulation of the TAM population.

Identification and characterization of a potent and selective HUNK inhibitor for treatment of HER2+ breast cancer.

Human epidermal growth factor receptor 2 (HER2)-targeted agents have proven to be effective, however, the development of resistance to these agents has become an obstacle in treating HER2+ breast cancer. Evidence implicates HUNK as an anti-cancer target for primary and resistant HER2+ breast cancers. In this study, a selective inhibitor of HUNK is characterized alongside a phosphorylation event in a downstream substrate of HUNK as a marker for HUNK activity in HER2+ breast cancer. Rubicon has been established as a substrate of HUNK that is phosphorylated at serine (S) 92. Findings indicate that HUNK-mediated phosphorylation of Rubicon at S92 promotes both autophagy and tumorigenesis in HER2/neu+ breast cancer. HUNK inhibition prevents Rubicon S92 phosphorylation in HER2/neu+ breast cancer models and inhibits tumorigenesis. This study characterizes a downstream phosphorylation event as a measure of HUNK activity and identifies a selective HUNK inhibitor that has meaningful efficacy toward HER2+ breast cancer.

HUNK Gene Alterations in Breast Cancer.

Hormonally upregulated neu-associated kinase (HUNK) is a serine/threonine (S/T) protein kinase related to the adenosine monophosphate-activated protein kinase (AMPK) family of kinases. HUNK was originally discovered using a screen to identify kinases expressed in the mouse mammary gland. Therefore, the majority of studies to date have been carried out in models specific to this tissue, and the kinase was named to reflect its mammary gland-specific physiology and pathology. Prior studies show a clear pathogenic role for HUNK in breast cancer. HUNK is upregulated in response to oncogenic HER2/neu and Akt, and there is strong evidence that HUNK is critical for the survival of breast cancer cells. Further evidence shows that inhibiting HUNK using a variety of breast cancer models, including those that are resistant, inhibits tumorigenesis and metastasis. However, HUNK alterations are infrequent. Here, the incidence and consequence of HUNK alterations in breast cancer is reviewed using data mined from the online database cBioPortal and considered in relation to prior research studies.

HUNK Signaling in Metastatic Breast Cancer.

Once metastatic disease has occurred, there is no cure for breast cancer. Consequently, identifying factors that promote and support breast cancer metastasis is critical for understanding how to pharmacologically target this process. Hormonally up-regulated neu-associated kinase (HUNK) is a serine/threonine protein kinase related to the sucrose non-fermenting-1 (Snf-1)/5' adenosine monophosphate-activated protein kinase (AMPK) family of kinases. HUNK has been found to play a role in breast cancer metastasis. However, conflicting reports indicate HUNK is a metastasis promoting factor as well as an inhibiting factor. Our group recently provided evidence that supports the conclusion that HUNK is a metastasis promoting factor by showing that HUNK regulates breast cancer metastasis through phosphorylation of EGFR. Here, we summarize our findings and discuss their implications toward pharmacological targeting of HUNK in breast cancer.

Staurosporine, an inhibitor of hormonally up-regulated neu-associated kinase.

HUNK is a protein kinase that is implicated in HER2-positive (HER2+) breast cancer progression and resistance to HER2 inhibitors. Though prior studies suggest there is therapeutic potential for targeting HUNK in HER2+ breast cancer, pharmacological agents that target HUNK are yet to be identified. A recent study showed that the broad-spectrum kinase inhibitor staurosporine binds to the HUNK catalytic domain, but the effect of staurosporine on HUNK enzymatic activity was not tested. We now show that staurosporine inhibits the kinase activity of a full length HUNK protein. Our findings further suggest that inhibiting HUNK with staurosporine has a strong effect on suppressing cell viability of HER2/neu mammary and breast cancer cells, which express high levels of HUNK protein and are dependent on HUNK for survival. Significantly, we use in vitro and in vivo methods to show that staurosporine synergizes with the HER2 inhibitor lapatinib to restore sensitivity toward HER2 inhibition in a HER2 inhibitor resistant breast cancer model. Collectively, these studies indicate that pharmacological inhibition of HUNK kinase activity has therapeutic potential for HER2+ breast cancers, including HER2+ breast cancers that have developed drug resistance.