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.

Inhibitory Effects of Alpha-Connexin Carboxyl-Terminal Peptide on Canine Mammary Epithelial Cells: A Study on Benign and Malignant Phenotypes.

Mammary cancer is highly prevalent in non-castrated female dogs. Cell-to-cell communication is an important mechanism to maintain homeostasis, and connexins are proteins that assemble to form the communicating gap junctions. In many cancers, communication capacity is reduced; several approaches are being tested in order to increase the communication capacity in cancer cells and, therefore, alter their viability. This study analyzed the effects of the alpha-connexin carboxyl-terminal peptide (αCT1) on canine mammary non-neoplastic and neoplastic epithelial cells. Seven canine epithelial mammary cell lines were used. Among these, one was a normal canine epithelial mammary cell line (LOEC-NMG), two canine mammary adenomas (LOEC-MAd1 and LOEC-MAd2), and four canine mammary adenocarcinomas (LOEC-MCA1, LOEC-MCA2, LOEC-MCA3 and CF41). The αCT1 corresponds to a short Cx43 C-terminal sequence linked to an internalization sequence called the antennapedia. After 24 h of incubation, the medium containing different αCT1 peptide concentrations was added to the cells, and only the culture medium was used for control. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was used to quantify cell viability before treatment and 48, 72, and 96 h after the treatment. Results showed that the normal mammary epithelial cell line (LOEC-NMG) was resistant to treatment with αCT1, which is consistent with a previous study on human mammary cell lines. One of the adenoma cell lines (LOEC-MAd2) was also resistant to treatment with αCT1, although the other (LOEC-MAd1) was susceptible to treatment, mostly at 72 h after treatment. Regarding the four canine adenocarcinoma cell lines, they differ regarding the susceptibility to the treatment with αCT1. Three cell lines, canine mixed adenocarcinoma (LOEC-MCA1), canine complex adenocarcinoma (LOEC-MCA2), and commercial canine mammary adenocarcinoma cell line CF41, were susceptible to treatment with αCT1, while one canine mammary adenocarcinoma cell line (LOEC-MCA3) was resistant to treatment. In most αCT1 treated cell lines, Cx43 was strongly detected in cell membranes by immunofluorescence. We propose that αCT1 restored the cell-to-cell communication capacity of neoplastic cells and induced inhibitory effects on cell viability.

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.

Special Issue: Cancer Metastasis and Therapeutic Resistance.

Metastasis and resistance to cancer therapeutics are critical barriers to curing cancer. This special issue entitled "Cancer Metastasis and Therapeutic Resistance" contains nine original contributions. The articles span a variety of human cancers, including breast, lung, brain, prostate, and skin and touch upon significant areas of interest such as cancer stem cell function, cancer immunology, and glycosylation.

Mass Spectrometry-Based Glycoproteomic Workflows for Cancer Biomarker Discovery.

Glycosylation has a clear role in cancer initiation and progression, with numerous studies identifying distinct glycan features or specific glycoproteoforms associated with cancer. Common findings include that aggressive cancers tend to have higher expression levels of enzymes that regulate glycosylation as well as glycoproteins with greater levels of complexity, increased branching, and enhanced chain length1. Research in cancer glycoproteomics over the last 50-plus years has mainly focused on technology development used to observe global changes in glycosylation. Efforts have also been made to connect glycans to their protein carriers as well as to delineate the role of these modifications in intracellular signaling and subsequent cell function. This review discusses currently available techniques utilizing mass spectrometry-based technologies used to study glycosylation and highlights areas for future advancement.

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.

Isoforms of Neuropilin-2 Denote Unique Tumor-Associated Macrophages in Breast Cancer.

Tumor-associated macrophages (TAMs) exert profound influence over breast cancer progression, promoting immunosuppression, angiogenesis, and metastasis. Neuropilin-2 (NRP2), consisting of the NRP2a and NRP2b isoforms, is a co-receptor for heparin-binding growth factors including VEGF-C and Class 3 Semaphorins. Selective upregulation in response to environmental stimuli and independent signaling pathways endow the NRP2 isoforms with unique functionality, with NRP2b promoting increased Akt signaling via receptor tyrosine kinases including VEGFRs, MET, and PDGFR. Although NRP2 has been shown to regulate macrophage/TAM biology, the role of the individual NRP2a/NRP2b isoforms in TAMs has yet to be evaluated. Using transcriptional profiling and spectral flow cytometry, we show that NRP2 isoform expression was significantly higher in TAMs from murine mammary tumors. NRP2a/NRP2b levels in human breast cancer metastasis were dependent upon the anatomic location of the tumor and significantly correlated with TAM infiltration in both primary and metastatic breast cancers. We define distinct phenotypes of NRP2 isoform-expressing TAMs in mouse models of breast cancer and within malignant pleural effusions from breast cancer patients which were exclusive of neuropilin-1 expression. Genetic depletion of either NRP2 isoform in macrophages resulted in a dramatic reduction of LPS-induced IL-10 production, defects in phagosomal processing of apoptotic breast cancer cells, and increase in cancer cell migration following co-culture. By contrast, depletion of NRP2b, but not NRP2a, inhibited production of IL-6. These results suggest that NRP2 isoforms regulate both shared and unique functionality in macrophages and are associated with distinct TAM subsets in breast cancer.

Effects of Alpha-Connexin Carboxyl-Terminal Peptide (aCT1) and Bowman-Birk Protease Inhibitor (BBI) on Canine Oral Mucosal Melanoma (OMM) Cells.

Oral mucosal melanomas (OMM) are aggressive cancers in dogs, and are good models for human OMM. Gap junctions are composed of connexin units, which may have altered expression patterns and/or subcellular localization in cancer cells. Cell-to-cell communication by gap junctions is often impaired in cancer cells, including in melanomas. Meanwhile, the upregulated expression of the gap junction protein connexin 43 (Cx43) inhibits melanoma progression. The α-connexin carboxyl-terminal (aCT1) peptide reportedly maintains Cx43 expression and cell-cell communication in human mammary cells and increases the communication activity through gap junctions in functional assays, therefore causing decreased cell proliferation. The Bowman-Birk protease inhibitor (BBI), a component of soybeans, induces Cx43 expression in several tumor cells as a trypsin-chymotrypsin inhibition function, with antineoplastic effects. This study investigated the effect of aCT1 peptide and BBI treatment, alone or in combination, on TLM1 canine melanoma cell viability. Cell viability after treatment with aCT1, the reverse sequence peptide (R-pep), and/or BBI for 5 days was analyzed by PrestoBlue assay. Immunofluorescence was used to observe Cx43 localization and expression. aCT1 (200 µM) alone did not significantly decrease cell viability in TLM1 cells, whereas BBI (400 µg/ml) alone significantly decreased the TLM1 viability. Combined treatment with both aCT1 (200 µM) and BBI (400 µg/ml) significantly decreased cell viability in TLM1 cells. Cx43 expression, as identified by immunostainings in TLM1 cells, was increased in the cell membrane after the combination treatment with BBI and aCT1. This dual treatment can be combined to achieve the anticancer activity, possibly by increasing Cx 43 expression and affecting Cx43 migration to the cell membrane. In conclusion, a treatment strategy targeting Cx43 with BBI and aCT1 may possibly lead to new effective therapies for canine OMM.

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 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.

Automating a Process Convolution Approach to Account for Spatial Information in Imaging Mass Spectrometry Data.

In the age of big data, imaging techniques such as imaging mass spectrometry (IMS) stand out due to the combination of data size and spatial referencing. However, the data analytic tools readily accessible to investigators often ignore the spatial information or provide results with vague interpretations. We focus on imaging techniques like IMS that collect data along a regular grid and develop methods to automate the process of modeling spatially-referenced imaging data using a process convolution (PC) approach. The PC approach provides a flexible framework to model spatially-referenced geostatistical data, but to make it computationally efficient requires identification of model parameters. We perform simulation studies to define optimal methods for specifying PC parameters and then test those methods using simulations that spike in real spatial information. In doing so, we demonstrate that our methods concurrently account for the spatial information and provide clear interpretations of covariate effects, while maximizing power and maintaining type I error rates near the nominal level. To make these methods accessible, we detail the imagingPC R package. Our approach provides a framework that is flexible and scalable to the level required by many imaging techniques.

Inhibition of the transcriptional kinase CDK7 overcomes therapeutic resistance in HER2-positive breast cancers.

Resistance of breast cancer to human epidermal growth factor receptor 2 (HER2) inhibitors involves reprogramming of the kinome through HER2/HER3 signaling via the activation of multiple tyrosine kinases and transcriptional upregulation. The heterogeneity of induced kinases prevents kinase targeting by a single kinase inhibitor and presents a major challenge to the treatment of therapeutically recalcitrant HER2-positive breast cancers (HER2+ BCs). As a result, there is a critical need for effective treatment that attacks the aberrant kinome activation associated with resistance to HER2-targeted therapy. Here, we describe a novel treatment strategy that targets cyclin-dependent kinase 7 (CDK7) in HER2 inhibitor-resistant (HER2iR) breast cancer. We show that both HER2 inhibitor-sensitive (HER2iS) and HER2iR breast cancer cell lines exhibit high sensitivity to THZ1, a newly identified covalent inhibitor of the transcription regulatory kinase CDK7. CDK7 promotes cell cycle progression through inhibition of transcription, rather than via direct phosphorylation of classical CDK targets. The transcriptional kinase activity of CDK7 is regulated by HER2, and by the receptor tyrosine kinases activated in response to HER2 inhibition, as well as by the downstream SHP2 and PI3K/AKT pathways. A low dose of THZ1 displayed potent synergy with the HER2 inhibitor lapatinib in HER2iR BC cells in vitro. Dual HER2 and CDK7 inhibition induced tumor regression in two HER2iR BC xenograft models in vivo. Our data support the utilization of CDK7 inhibition as an additional therapeutic avenue that blocks the activation of genes engaged by multiple HER2iR kinases.

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.

Polo-like kinase 1 (Plk1) inhibition synergizes with taxanes in triple negative breast cancer.

Within triple negative breast cancer, several molecular subtypes have been identified, underlying the heterogeneity of such an aggressive disease. The basal-like subtype is characterized by mutations in the TP53 gene, and is associated with a low pathologic complete response rate following neoadjuvant chemotherapy. In a genome-scale short hairpin RNA (shRNA) screen of breast cancer cells, polo-like kinase 1 (Plk1) was a frequent and strong hit in the basal breast cancer cell lines indicating its importance for growth and survival of these breast cancer cells. Plk1 regulates progression of cells through the G2-M phase of the cell cycle. We assessed the activity of two ATP-competitive Plk1 inhibitors, GSK461364 and onvansertib, alone and with a taxane in a set of triple negative breast cancer cell lines and in vivo. GSK461364 showed synergism with docetaxel in SUM149 (Combination Index 0.70) and SUM159 (CI, 0.62). GSK461364 in combination with docetaxel decreased the clonogenic potential (interaction test for SUM149 and SUM159, p<0.001 and p = 0.01, respectively) and the tumorsphere formation of SUM149 and SUM159 (interaction test, p = 0.01 and p< 0.001). In the SUM159 xenograft model, onvansertib plus paclitaxel significantly decreased tumor volume compared to single agent paclitaxel (p<0.0001). Inhibition of Plk1 in combination with taxanes shows promising results in a subset of triple negative breast cancer intrinsically resistant to chemotherapy. Onvansertib showed significant tumor volume shrinkage when combined with paclitaxel in vivo and should be considered in clinical trials for the treatment of triple negative cancers.

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.

A Rapid Array-Based Approach to N-Glycan Profiling of Cultured Cells.

Typically, N-glycosylation studies done on cultured cells require up to millions of cells followed by lengthy preparation to release, isolate, and profile N-glycans. To overcome these limitations, we report a rapid array-based workflow for profiling N-glycan signatures from cells, adapted from imaging mass spectrometry used for on-tissue N-glycan profiling. Using this approach, N-glycan profiles from a low-density array of eight cell chambers could be reported within 4 h of completing cell culture. Approaches are demonstrated that account for background N-glycans due to serum media. Normalization procedures are shown. The method is robust and reproducible, requiring as few as 3000 cells per replicate with a 3-20% coefficient of variation to capture label-free profiles of N-glycans. Quantification by stable isotopic labeling of N-glycans in cell culture is demonstrated and adds no additional time to preparation. Utility of the method is demonstrated by measurement of N-glycan turnover rates due to induction of oxidative stress in human primary aortic endothelial cells. The developed method and ancillary tools serve as a foundational launching point for rapid profiling of N-glycans ranging from high-density arrays down to single cells in culture.

Uncoupling of p97 ATPase activity has a dominant negative effect on protein extraction.

p97 is a highly abundant, homohexameric AAA+ ATPase that performs a variety of essential cellular functions. Characterized as a ubiquitin-selective chaperone, p97 recognizes proteins conjugated to K48-linked polyubiquitin chains and promotes their removal from chromatin and other molecular complexes. Changes in p97 expression or activity are associated with the development of cancer and several related neurodegenerative disorders. Although pathogenic p97 mutations cluster in and around p97's ATPase domains, mutant proteins display normal or elevated ATPase activity. Here, we show that one of the most common p97 mutations (R155C) retains ATPase activity, but is functionally defective. p97-R155C can be recruited to ubiquitinated substrates on chromatin, but is unable to promote substrate removal. As a result, p97-R155C acts as a dominant negative, blocking protein extraction by a similar mechanism to that observed when p97's ATPase activity is inhibited or inactivated. However, unlike ATPase-deficient proteins, p97-R155C consumes excess ATP, which can hinder high-energy processes. Together, our results shed new insight into how pathogenic mutations in p97 alter its cellular function, with implications for understanding the etiology and treatment of p97-associated diseases.