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.

Mapping the Anti-Cancer Activity of α-Connexin Carboxyl-Terminal (aCT1) Peptide in Resistant HER2+ Breast Cancer.

Connexin 43 (Cx43) is a protein encoded by the GJA1 gene and is a component of cell membrane structures called gap junctions, which facilitate intercellular communication. Prior evidence indicates that elevated GJA1 expression in the HER2-positive (HER2+) subtype of breast cancer is associated with poor prognosis. Prior evidence also suggests that HER2+ breast cancers that have become refractory to HER2-targeted agents have a loss of Cx43 gap junction intercellular communication (GJIC). In this study, a Cx43-targeted agent called alpha-connexin carboxyl-terminal peptide (aCT1) is examined to determine whether GJIC can be rescued in refractory HER2+ breast cancer cells. A proposed mechanism of action for aCT1 is binding to the tight junction protein Zonal Occludens-1 (ZO-1). However, the true scope of activity for aCT1 has not been explored. In this study, mass spectrometry proteomic analysis is used to determine the breadth of aCT1-interacting proteins. The NanoString nCounter Breast Cancer 360 panel is also used to examine the effect of aCT1 on cancer signaling in HER2+ breast cancer cells. Findings from this study show a dynamic range of binding partners for aCT1, many of which regulate gene expression and RNA biology. nCounter analysis shows that a number of pathways are significantly impacted by aCT1, including upregulation of apoptotic factors, leading to the prediction and demonstration that aCT1 can boost the cell death effects of cisplatin and lapatinib in HER2+ breast cancer cells that have become resistant to HER2-targeted agents.

NF-κB Signaling Is Regulated by Fucosylation in Metastatic Breast Cancer Cells.

A growing body of evidence indicates that the levels of fucosylation correlate with breast cancer progression and contribute to metastatic disease. However, very little is known about the signaling and functional outcomes that are driven by fucosylation. We performed a global proteomic analysis of 4T1 metastatic mammary tumor cells in the presence and absence of a fucosylation inhibitor, 2-fluorofucose (2FF). Of significant interest, pathway analysis based on our results revealed a reduction in the NF-κB and TNF signaling pathways, which regulate the inflammatory response. NF-κB is a transcription factor that is pro-tumorigenic and a prime target in human cancer. We validated our results, confirming that treatment of 4T1 cells with 2FF led to a decrease in NF-κB activity through increased IκBα. Based on these observations, we conclude that fucosylation is an important post-translational modification that governs breast cancer cell signaling.

HUNK Phosphorylates Rubicon to Support Autophagy.

BACKGROUND: Autophagy is a catabolic cellular recycling pathway that is essential for maintaining intracellular homeostasis. Autophagosome formation is achieved via the coordination of the Beclin-1 protein complex. Rubicon is a Beclin-1 associated protein that suppresses autophagy by impairing the activity of the class III PI3K, Vps34. However, very little is known about the molecular mechanisms that regulate Rubicon function. METHODS: In this study, co-immunoprecipitation and kinase assays were used to investigate the ability of Hormonally Upregulated Neu-associated Kinase (HUNK) to bind to and phosphorylate Rubicon. LC3B was monitored by immunofluorescence and immunoblotting to determine whether phosphorylation of Rubicon by HUNK controls the autophagy suppressive function of Rubicon. RESULTS: Findings from this study identify Rubicon as a novel substrate of HUNK and show that phosphorylation of Rubicon inhibits its function, promoting autophagy.

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

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 in vivo.

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR.

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death. Common sites of metastatic spread include lung, lymph node, brain, and bone. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

Targeting connexin 43 with α-connexin carboxyl-terminal (ACT1) peptide enhances the activity of the targeted inhibitors, tamoxifen and lapatinib, in breast cancer: clinical implication for ACT1.

BACKGROUND: Treatment failure is a critical issue in breast cancer and identifying useful interventions that optimize current cancer therapies remains a critical unmet need. Expression and functional studies have identified connexins (Cxs), a family of gap junction proteins, as potential tumor suppressors. Studies suggest that Cx43 has a role in breast cancer cell proliferation, differentiation, and migration. Although pan-gap junction drugs are available, the lack of specificity of these agents increases the opportunity for off target effects. Consequently, a therapeutic agent that specifically modulates Cx43 would be beneficial and has not been tested in breast cancer. In this study, we now test an agent that specifically targets Cx43, called ACT1, in breast cancer. METHODS: We evaluated whether direct modulation of Cx43 using a Cx43-directed therapeutic peptide, called ACT1, enhances Cx43 gap junctional activity in breast cancer cells, impairs breast cancer cell proliferation or survival, and enhances the activity of the targeted inhibitors tamoxifen and lapatinib. RESULTS: Our results show that therapeutic modulation of Cx43 by ACT1 maintains Cx43 at gap junction sites between cell-cell membrane borders of breast cancer cells and augments gap junction activity in functional assays. The increase in Cx43 gap junctional activity achieved by ACT1 treatment impairs proliferation or survival of breast cancer cells but ACT1 has no effect on non-transformed MCF10A cells. Furthermore, treating ER+ breast cancer cells with a combination of ACT1 and tamoxifen or HER2+ breast cancer cells with ACT1 and lapatinib augments the activity of these targeted inhibitors. CONCLUSIONS: Based on our findings, we conclude that modulation of Cx43 activity in breast cancer can be effectively achieved with the agent ACT1 to sustain Cx43-mediated gap junctional activity resulting in impaired malignant progression and enhanced activity of lapatinib and tamoxifen, implicating ACT1 as part of a combination regimen in breast cancer.

Regulation of cell survival by HUNK mediates breast cancer resistance to HER2 inhibitors.

Breast cancer patients who are HER2-positive receive targeted inhibitors to HER2, including trastuzumab and lapatinib. While patients benefit from the use of HER2 inhibitors, many fail therapy and almost all patients become resistant to treatment, indicating a critical need to prevent treatment failure. Several recent studies indicate that activation of autophagy contributes to trastuzumab and lapatinib resistance and demonstrate that impairing autophagy in breast cancer cells is therapeutically beneficial. Moreover, autophagy is mechanistically linked through signaling crosstalk to apoptotic pathways, where activation of one process impacts the other. Therefore, understanding the molecular mechanisms that control these processes may uncover novel areas of therapeutic intervention to combat or prevent resistance in breast cancer. We previously characterized the protein kinase HUNK as a breast cancer-promoting factor in HER2/neu-induced mammary tumor models, in which HUNK supported the survival of HER2/neu-positive tumor cells, likely through the regulation of apoptosis. Because significant crosstalk exists between apoptotic and autophagy proteins, we now examine if HUNK is also able to regulate cell survival through modulation of autophagy using HER2 inhibitor sensitive and resistant breast cancer models. Furthermore, we investigate whether inhibiting HUNK impairs in vivo tumor growth that is initiated by HER2 inhibitor-resistant breast cancer cells. Our findings indicate that therapeutically targeting HUNK is a potential strategy for overcoming resistance and that resistant breast cancer cells maintain HUNK expression to drive tumorigenesis, an observation that is consistent with a pro-survival role for this kinase.