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.

Toll-Like Receptor 3 Deficiency Leads to Altered Immune Responses to Chlamydia trachomatis Infection in Human Oviduct Epithelial Cells.

Reproductive tract pathology caused by Chlamydia trachomatis infection is an important global cause of human infertility. To better understand the mechanisms associated with Chlamydia-induced genital tract pathogenesis in humans, we used CRISPR genome editing to disrupt Toll-like receptor 3 (TLR3) function in the human oviduct epithelial (hOE) cell line OE-E6/E7 in order to investigate the possible role(s) of TLR3 signaling in the immune response to Chlamydia Disruption of TLR3 function in these cells significantly diminished the Chlamydia-induced synthesis of several inflammation biomarkers, including interferon beta (IFN-ß), interleukin-6 (IL-6), interleukin-6 receptor alpha (IL-6Rα), soluble interleukin-6 receptor beta (sIL-6Rß, or gp130), IL-8, IL-20, IL-26, IL-34, soluble tumor necrosis factor receptor 1 (sTNF-R1), tumor necrosis factor ligand superfamily member 13B (TNFSF13B), matrix metalloproteinase 1 (MMP-1), MMP-2, and MMP-3. In contrast, the Chlamydia-induced synthesis of CCL5, IL-29 (IFN-λ1), and IL-28A (IFN-λ2) was significantly increased in TLR3-deficient hOE cells compared to their wild-type counterparts. Our results indicate a role for TLR3 signaling in limiting the genital tract fibrosis, scarring, and chronic inflammation often associated with human chlamydial disease. Interestingly, we saw that Chlamydia infection induced the production of biomarkers associated with persistence, tumor metastasis, and autoimmunity, such as soluble CD163 (sCD163), chitinase-3-like protein 1, osteopontin, and pentraxin-3, in hOE cells; however, their expression levels were significantly dysregulated in TLR3-deficient hOE cells. Finally, we demonstrate using hOE cells that TLR3 deficiency resulted in an increased amount of chlamydial lipopolysaccharide (LPS) within Chlamydia inclusions, which is suggestive that TLR3 deficiency leads to enhanced chlamydial replication and possibly increased genital tract pathogenesis during human infection.

TLR3 deficiency exacerbates the loss of epithelial barrier function during genital tract Chlamydia muridarum infection.

PROBLEM: Chlamydia trachomatis infections are often associated with acute syndromes including cervicitis, urethritis, and endometritis, which can lead to chronic sequelae such as pelvic inflammatory disease (PID), chronic pelvic pain, ectopic pregnancy, and tubal infertility. As epithelial cells are the primary cell type productively infected during genital tract Chlamydia infections, we investigated whether Chlamydia has any impact on the integrity of the host epithelial barrier as a possible mechanism to facilitate the dissemination of infection, and examined whether TLR3 function modulates its impact. METHOD OF STUDY: We used wild-type and TLR3-deficient murine oviduct epithelial (OE) cells to ascertain whether C. muridarum infection had any effect on the epithelial barrier integrity of these cells as measured by transepithelial resistance (TER) and cell permeability assays. We next assessed whether infection impacted the transcription and protein function of the cellular tight-junction (TJ) genes for claudins1-4, ZO-1, JAM1 and occludin via quantitative real-time PCR (qPCR) and western blot. RESULTS: qPCR, immunoblotting, transwell permeability assays, and TER studies show that Chlamydia compromises cellular TJ function throughout infection in murine OE cells and that TLR3 deficiency significantly exacerbates this effect. CONCLUSION: Our data show that TLR3 plays a role in modulating epithelial barrier function during Chlamydia infection of epithelial cells lining the genital tract. These findings propose a role for TLR3 signaling in maintaining the integrity of epithelial barrier function during genital tract Chlamydia infection, a function that we hypothesize is important in helping limit the chlamydial spread and subsequent genital tract pathology.