(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 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.

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.

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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.

Human epidermal growth factor receptor 2‐positive (HER2+) breast cancer is one of the three clinical subtypes of breast cancer and is defined by having HER2 gene amplification that coincides with HER2 protein overexpression. HER2 is amplified in 15‐30% of breast cancers and overexpression of this gene is a predictor of survival in breast cancer patients. HER2‐targeted therapies have been successful in treating HER2+ breast cancer; however, over time, HER2+ breast cancer can develop resistance to these therapies. Therefore, there is an urgent need to establish novel targeted agents. One proposed method of resistance to these HER2 agents is dysregulation of autophagy where cancer cells can shift to a cytoprotective autophagy phenotype to avoid cell death. Previous work in our lab established that Hormonally Upregulated Neu‐associated kinase (HUNK) is a Serine/Threonine (S/T) protein kinase that is overexpressed in HER2+ breast cancer and is responsible for promoting autophagy, thereby leading to therapeutic resistance. Our previous work shows that HUNK phosphorylates an autophagy protein, Rubicon, in the N‐terminal domain at S92, promoting autophagy in 293T cells. However, we have yet to establish a role for this phosphorylation site within HER2+ breast cancer cells. Therefore, the objective of this study is to test whether Rubicon S92 phosphorylation plays a role in promoting tumorigenesis in HER2+ breast cancer. We generated multiple scientific tools: a phosphoserine antibody at S92 in Rubicon, a phospho‐mimetic (S92A) mutant, and a phospho‐deficient (S92D) mutant. We have established an autophagy phenotype within a HER2/neu model; mouse mammary tumor virus driven neu(rodent form of HER2) expressing mammary tumor cells (MMTV‐neu) genetically engineered to be HUNK wild type (WT) or HUNK knockout (KO). We performed an endogenous Rubicon immunoprecipitation (IP) and found that KO cells are deficient in S92 phosphorylation alongside with a lack of autophagy response, measured using LC3‐II western blot. These findings confirm that HUNK is required for Rubicon S92 phosphorylation in a genetically tractable model. Utilizing these cell lines alongside the phospho‐mimetic and phospho‐deficient Rubicon mutants will allow us to determine the role that HUNK plays in promoting tumorigenesis within HER2+ breast cancer in‐vitro and in‐vivo.