Keratin 17 modulates the immune topography of pancreatic cancer.

BACKGROUND: The immune microenvironment impacts tumor growth, invasion, metastasis, and patient survival and may provide opportunities for therapeutic intervention in pancreatic ductal adenocarcinoma (PDAC). Although never studied as a potential modulator of the immune response in most cancers, Keratin 17 (K17), a biomarker of the most aggressive (basal) molecular subtype of PDAC, is intimately involved in the histogenesis of the immune response in psoriasis, basal cell carcinoma, and cervical squamous cell carcinoma. Thus, we hypothesized that K17 expression could also impact the immune cell response in PDAC, and that uncovering this relationship could provide insight to guide the development of immunotherapeutic opportunities to extend patient survival. METHODS: Multiplex immunohistochemistry (mIHC) and automated image analysis based on novel computational imaging technology were used to decipher the abundance and spatial distribution of T cells, macrophages, and tumor cells, relative to K17 expression in 235 PDACs. RESULTS: K17 expression had profound effects on the exclusion of intratumoral CD8+ T cells and was also associated with decreased numbers of peritumoral CD8+ T cells, CD16+ macrophages, and CD163+ macrophages (p < 0.0001). The differences in the intratumor and peritumoral CD8+ T cell abundance were not impacted by neoadjuvant therapy, tumor stage, grade, lymph node status, histologic subtype, nor KRAS, p53, SMAD4, or CDKN2A mutations. CONCLUSIONS: Thus, K17 expression correlates with major differences in the immune microenvironment that are independent of any tested clinicopathologic or tumor intrinsic variables, suggesting that targeting K17-mediated immune effects on the immune system could restore the innate immunologic response to PDAC and might provide novel opportunities to restore immunotherapeutic approaches for this most deadly form of cancer.

Neutralizing activity to SARS-CoV-2 of convalescent and control plasma used in a randomized controlled trial.

BACKGROUND: There are limited data on the neutralizing activity of convalescent plasma (CP) administered in randomized controlled trials (RCT) of COVID-19 infection. STUDY DESIGN AND METHODS: As part of an RCT, CP was collected per FDA guidelines from individuals recovered from COVID-19 infection. CP donors had to have ≥145 optical density (OD) units (ideal target ≥300) using a semiquantitative, immunochromatographic test for IgG antibody to the nucleocapsid protein (NP) of SARS-CoV-2 (typical range 0-500 OD units). A random subset of samples [14 control plasma, 12 CP "medium-anti-NP" (145-299 OD units), and 13 CP "high" anti-NP (≥300 OD units)] were tested for neutralizing antibodies using an established viral luciferase antibody inhibition assay to detect the infection of SARS-CoV-2 pseudovirus that encoded spike protein (SARS2-Strunc ) on a human immunodeficiency virus 1 vector (NL43dEnvNanoLuc), using ACE2-expressing 293 T cells. The titer needed to neutralize 50% of viral activity (NT50) was calculated. RESULTS: The uptake of SARS-CoV-2 pseudovirus by 293TACE2 cells was inhibited by pretreatment with CP compared to control CP (p < .001) with control plasma having a median (IQR) 50% neutralization titer (NT50) of 1:28 (1:16,1:36) compared to 1:334 (1:130,1:1295) and 1:324 (1:244,1:578), for medium anti-NP and high anti-NP CP units, respectively. The neutralizing activity of CP met minimum FDA criteria with neutralizing antibody titers >1:80 in 100% of randomly selected samples, using a conservative approach that excluded non-specific binding. DISCUSSION: Plasma from donors screened using an immunochromatographic test for IgG antibody to SARS-CoV-2 NP exhibited neutralizing activity meeting FDA's minimum standard in all randomly selected COVID-19 CP units.

The extracellular matrix protein laminin α2 regulates the maturation and function of the blood-brain barrier.

Laminins are major constituents of the gliovascular basal lamina of the blood-brain barrier (BBB); however, the role of laminins in BBB development remains unclear. Here we report that Lama2(-/-) mice, lacking expression of the laminin α2 subunit of the laminin-211 heterotrimer expressed by astrocytes and pericytes, have a defective BBB in which systemically circulated tracer leaks into the brain parenchyma. The Lama2(-/-) vascular endothelium had significant abnormalities, including altered integrity and composition of the endothelial basal lamina, inappropriate expression of embryonic vascular endothelial protein MECA32, substantially reduced pericyte coverage, and tight junction abnormalities. Additionally, astrocytic endfeet were hypertrophic and lacked appropriately polarized aquaporin4 channels. Laminin-211 appears to mediate these effects at least in part by dystroglycan receptor interactions, as preventing dystroglycan expression in neural cells led to a similar set of BBB abnormalities and gliovascular disturbances, which additionally included perturbed vascular endothelial glucose transporter-1 localization. These findings provide insight into the cell and molecular changes that occur in congenital muscular dystrophies caused by Lama2 mutations or inappropriate dystroglycan post-translational modifications, which have accompanying brain abnormalities, including seizures. Our results indicate a novel role for laminin-dystroglycan interactions in the cooperative integration of astrocytes, endothelial cells, and pericytes in regulating the BBB.

Laminin promotes metalloproteinase-mediated dystroglycan processing to regulate oligodendrocyte progenitor cell proliferation.

The cell surface receptor dystroglycan mediates interactions between oligodendroglia and laminin-211, an extracellular matrix protein that regulates timely oligodendroglial development. However, dystroglycan's precise role in oligodendroglial development and the potential mechanisms to regulate laminin-dystroglycan interactions remain unknown. Here we report that oligodendroglial dystroglycan is cleaved by metalloproteinases, thereby uncoupling oligodendroglia from laminin binding. Dystroglycan cleavage is selectively stimulated by oligodendrocyte progenitor cell attachment to laminin-211, but not laminin-111 or poly-D-lysine. In addition, dystroglycan cleavage occurs most prominently in oligodendrocyte progenitor cells, with limited dystroglycan cleavage observed in differentiating oligodendrocytes. When dystroglycan cleavage is blocked by metalloproteinase inhibitors, oligodendrocyte progenitor cell proliferation is substantially decreased. Conversely, expression of the intracellular portion of cleaved dystroglycan results in increased oligodendrocyte progenitor cell proliferation, suggesting that endogenous dystroglycan cleavage may promote oligodendrocyte progenitor cell cycle progression. Intriguingly, while matrix metalloproteinase-2 and/or -9 have been reported to be responsible for dystroglycan cleavage, we find that these two metalloproteinases are neither necessary nor sufficient for cleavage of oligodendroglial dystroglycan. In summary, laminin-211 stimulates metalloproteinase-mediated dystroglycan cleavage in oligodendrocyte progenitor cells (but not in differentiated oligodendrocytes), which in turn promotes oligodendrocyte progenitor cell proliferation. This novel regulation of oligodendroglial laminin-dystroglycan interactions may have important consequences for oligodendroglial differentiation, both during development and during disease when metalloproteinase levels become elevated.

Keratin 17 modulates the immune topography of pancreatic cancer.

Background: The immune microenvironment impacts tumor growth, invasion, metastasis, and patient survival and may provide opportunities for therapeutic intervention in pancreatic ductal adenocarcinoma (PDAC). Although never studied as a potential modulator of the immune response in most cancers, Keratin 17 (K17), a biomarker of the most aggressive (basal) molecular subtype of PDAC, is intimately involved in the histogenesis of the immune response in psoriasis, basal cell carcinoma, and cervical squamous cell carcinoma. Thus, we hypothesized that K17 expression could also impact the immune cell response in PDAC, and that uncovering this relationship could provide insight to guide the development of immunotherapeutic opportunities to extend patient survival. Methods: Multiplex immunohistochemistry (mIHC) and automated image analysis based on novel computational imaging technology were used to decipher the abundance and spatial distribution of T cells, macrophages, and tumor cells, relative to K17 expression in 235 PDACs. Results: K17 expression had profound effects on the exclusion of intratumoral CD8 + T cells and was also associated with decreased numbers of peritumoral CD8 + T cells, CD16 + macrophages, and CD163 + macrophages (p < 0.0001). The differences in the intratumor and peritumoral CD8 + T cell abundance were not impacted by neoadjuvant therapy, tumor stage, grade, lymph node status, histologic subtype, nor KRAS, p53, SMAD4, or CDKN2A mutations. Conclusions: Thus, K17 expression correlates with major differences in the immune microenvironment that are independent of any tested clinicopathologic or tumor intrinsic variables, suggesting that targeting K17-mediated immune effects on the immune system could restore the innate immunologic response to PDAC and might provide novel opportunities to restore immunotherapeutic approaches for this most deadly form of cancer.

Dissecting the Oncogenic Roles of Keratin 17 in the Hallmarks of Cancer.

There is an unmet need to identify and validate tumor-specific therapeutic targets to enable more effective treatments for cancer. Heterogeneity in patient clinical characteristics as well as biological and genetic features of tumors present major challenges for the optimization of therapeutic interventions, including the development of novel and more effective precision medicine. The expression of keratin 17 (K17) is a hallmark of the most aggressive forms of cancer across a wide range of anatomical sites and histological types. K17 correlates with shorter patient survival, predicts resistance to specific chemotherapeutic agents, and harbors functional domains that suggest it could be therapeutically targeted. Here, we explore the role of K17 in the hallmarks of cancer and summarize evidence to date for K17-mediated mechanisms involved in each hallmark, elucidating functional roles that warrant further investigation to guide the development of novel therapeutic strategies.

Keratin 17 testing in pancreatic cancer needle aspiration biopsies predicts survival.

BACKGROUND: Although pancreatic ductal adenocarcinoma (PDAC) has one of the lowest 5-year survival rates of all cancers, differences in survival exist between patients with clinically identical characteristics. The authors previously demonstrated that keratin 17 (K17) expression in PDAC, measured by RNA sequencing or immunohistochemistry (IHC), is an independent negative prognostic biomarker. Only 20% of cases are candidates for surgical resection, but most patients are diagnosed by needle aspiration biopsy (NAB). The aims of this study were to determine whether there was a correlation in K17 scores detected in matched NABs and surgical resection tissue sections and whether K17 IHC in NAB cell block specimens could be used as a negative prognostic biomarker in PDAC. METHODS: K17 IHC was performed for a cohort of 70 patients who had matched NAB cell block and surgical resection samples to analyze the correlation of K17 expression levels. K17 IHC was also performed in cell blocks from discovery and validation cohorts. Kaplan-Meier and Cox proportional hazards regression models were analyzed to determine survival differences in cases with different levels of K17 IHC expression. RESULTS: K17 IHC expression correlated in matched NABs and resection tissues. NAB samples were classified as high for K17 when ≥80% of tumor cells showed strong (2+) staining. High-K17 cases, including stage-matched cases, had shorter survival. CONCLUSIONS: K17 has been identified as a robust and independent prognostic biomarker that stratifies clinical outcomes for cases that are diagnosed by NAB. Testing for K17 also has the potential to inform clinical decisions for optimization of chemotherapeutic interventions.

Tyrosine phosphatases Shp1 and Shp2 have unique and opposing roles in oligodendrocyte development.

Oligodendrocyte progenitor cells first proliferate to generate sufficient cell numbers and then differentiate into myelin-producing oligodendrocytes. The signal transduction mediators that underlie these events, however, remain poorly understood. The tyrosine phosphatase Shp1 has been linked to oligodendrocyte differentiation as Shp1-deficient mice show hypomyelination. The Shp1 homolog, Shp2, has recently been shown to regulate astrogliogenesis, but its role in oligodendrocyte development remains unknown. Here, we report that Shp2 protein levels were developmentally regulated in oligodendrocytes, with Shp2 phosphorylation being promoted by oligodendroglial mitogens but suppressed by laminin, an extracellular matrix protein that promotes oligodendroglial differentiation. In contrast, oligodendrocyte progenitors were found to be unresponsive to mitogens following Shp2, but not Shp1, depletion. In agreement with previous studies, Shp1 depletion led to decreased levels of myelin basic protein in differentiating oligodendrocytes, as well as reduced outgrowth of myelin membrane sheets. Shp2 depletion in contrast did not prevent oligodendrocyte differentiation but promoted expanded myelin membrane outgrowth. Taken together these data suggest that Shp1 and Shp2 have distinct functions in oligodendrocyte development: Shp2 regulates oligodendrocyte progenitor proliferation and Shp1 regulates oligodendrocyte differentiation. Adhesion to laminin may additionally provide extrinsic regulation of Shp2 activity and thus promote the transition from progenitor to differentiating oligodendrocyte.

An unbiased high-throughput drug screen reveals a potential therapeutic vulnerability in the most lethal molecular subtype of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is predicted to become the second leading cause of cancer-related deaths in the United States by 2020, due in part to innate resistance to widely used chemotherapeutic agents and limited knowledge about key molecular factors that drive tumor aggression. We previously reported a novel negative prognostic biomarker, keratin 17 (K17), whose overexpression in cancer results in shortened patient survival. In this study, we aimed to determine the predictive value of K17 and explore the therapeutic vulnerability in K17-expressing PDAC, using an unbiased high-throughput drug screen. Patient-derived data analysis showed that K17 expression correlates with resistance to gemcitabine (Gem). In multiple in vitro and in vivo models of PDAC, spanning human and murine PDAC cells, and orthotopic xenografts, we determined that the expression of K17 results in a more than twofold increase in resistance to Gem and 5-fluorouracil, key components of current standard-of-care chemotherapeutic regimens. Furthermore, through an unbiased drug screen, we discovered that podophyllotoxin (PPT), a microtubule inhibitor, showed significantly higher sensitivity in K17-positive compared to K17-negative PDAC cell lines and animal models. In the clinic, another microtubule inhibitor, paclitaxel (PTX), is used in combination with Gem as a first-line chemotherapeutic regimen for PDAC. Surprisingly, we found that when combined with Gem, PPT, but not PTX, was synergistic in inhibiting the viability of K17-expressing PDAC cells. Importantly, in preclinical models, PPT in combination with Gem effectively decreased tumor growth and enhanced the survival of mice bearing K17-expressing tumors. This provides evidence that PPT and its derivatives could potentially be combined with Gem to enhance treatment efficacy for the ~ 50% of PDACs that express high levels of K17. In summary, we reported that K17 is a novel target for developing a biomarker-based personalized treatment for PDAC.

Keratin 17 identifies the most lethal molecular subtype of pancreatic cancer.

Although the overall five-year survival of patients with pancreatic ductal adenocarcinoma (PDAC) is dismal, there are survival differences between cases with clinically and pathologically indistinguishable characteristics, suggesting that there are uncharacterized properties that drive tumor progression. Recent mRNA sequencing studies reported gene-expression signatures that define PDAC molecular subtypes that correlate with differences in survival. We previously identified Keratin 17 (K17) as a negative prognostic biomarker in other cancer types. Here, we set out to determine if K17 is as accurate as molecular subtyping of PDAC to identify patients with the shortest survival. K17 mRNA was analyzed in two independent PDAC cohorts for discovery (n = 124) and validation (n = 145). Immunohistochemical localization and scoring of K17 immunohistochemistry (IHC) was performed in a third independent cohort (n = 74). Kaplan-Meier and Cox proportional-hazard regression models were analyzed to determine cancer specific survival differences in low vs. high mRNA K17 expressing cases. We established that K17 expression in PDACs defines the most aggressive form of the disease. By using Cox proportional hazard ratio, we found that increased expression of K17 at the IHC level is also associated with decreased survival of PDAC patients. Additionally, within PDACs of advanced stage and negative surgical margins, K17 at both mRNA and IHC level is sufficient to identify the subgroup with the shortest survival. These results identify K17 as a novel negative prognostic biomarker that could inform patient management decisions.

Astrocyte HIF-2α supports learning in a passive avoidance paradigm under hypoxic stress.

BACKGROUND: The brain is extensively vascularized, uses20% of the body's oxygen, and is highly sensitive to changes in oxygen. While synaptic plasticity and memory are impaired in healthy individuals by exposure to mild hypoxia, aged individuals appear to be even more sensitive. Aging is associated with progressive failure in pulmonary and cardiovascular systems, exposing the aged to both chronic and superimposed acute hypoxia. The HIF proteins, the "master regulators" of the cellular response to hypoxia, are robustly expressed in neurons and astrocytes. Astrocytes support neurons and synaptic plasticity via complex metabolic and trophic mechanisms. The activity of HIF proteins in the brain is diminished with aging, and the increased exposure to chronic and acute hypoxia with aging combined with diminished HIF activity may impair synaptic plasticity. PURPOSE: Herein, we test the hypothesis that astrocyte HIF supports synaptic plasticity and learning upon hypoxia. MATERIALS AND METHODS: An Astrocyte-specific HIF loss-of-function model was employed, where knock-out of HIF-1α or HIF-2α in GFAP expressing cells was accomplished by cre-mediated recombination. Animals were tested for behavioral (open field and rotarod), learning (passive avoidance paradigm), and electrophysiological (long term potentiation) responses to mild hypoxic challenge. RESULTS: In an astrocyte-specific HIF loss-of-function model followed by mild hypoxia, we identified that the depletion of HIF-2α resulted in an impaired passive avoidance learning performance. This was accompanied by an attenuated response to induction in long-term potentiation (LTP), suggesting that the hippocampal circuitry was perturbed upon hypoxic exposure following HIF-2α loss in astrocytes, and not due to hippocampal cell death. We investigated HIF-regulated trophic and metabolic target genes and found that they were not regulated by HIF-2α, suggesting that these specific targets may not be involved in mediating the phenotypes observed. CONCLUSION: Together, these results point to a role for HIF-2α in the astrocyte's regulatory role in synaptic plasticity and learning under hypoxia and suggest that even mild, acute hypoxic challenges can impair cognitive performance in the aged population who harbor impaired HIF function.

Behavioral experiences as drivers of oligodendrocyte lineage dynamics and myelin plasticity.

Many behavioral experiences are known to promote hippocampal neurogenesis. In contrast, the ability of behavioral experiences to influence the production of oligodendrocytes and myelin sheath formation remains relatively unknown. However, several recent studies indicate that voluntary exercise and environmental enrichment can positively influence both oligodendrogenesis and myelination, and that, in contrast, social isolation can negatively influence myelination. In this review we summarize studies addressing the influence of behavioral experiences on oligodendrocyte lineage cells and myelin, and highlight potential mechanisms including experience-dependent neuronal activity, metabolites, and stress effectors, as well as both local and systemic secreted factors. Although more study is required to better understand the underlying mechanisms by which behavioral experiences regulate oligodendrocyte lineage cells, this exciting and newly emerging field has already revealed that oligodendrocytes and their progenitors are highly responsive to behavioral experiences and suggest the existence of a complex network of reciprocal interactions among oligodendrocyte lineage development, behavioral experiences, and brain function. Achieving a better understanding of these relationships may have profound implications for human health, and in particular, for our understanding of changes in brain function that occur in response to experiences. This article is part of the Special Issue entitled 'Oligodendrocytes in Health and Disease'.