Genomic epidemiology reveals the dominance of Hennepin County in the transmission of SARS-CoV-2 in Minnesota from 2020 to 2022.

IMPORTANCE: We analyzed over 22,000 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes of patient samples tested at Mayo Clinic Laboratories during a 2-year period in the COVID-19 pandemic, which included Alpha, Delta, and Omicron variants of concern to examine the roles and relationships of Minnesota virus transmission. We found that Hennepin County, the most populous county, drove the transmission of SARS-CoV-2 viruses in the state after including the formation of earlier clades including 20A, 20C, and 20G, as well as variants of concern Alpha and Delta. We also found that Hennepin County was the source for most of the county-to-county introductions after an initial predicted introduction with the virus in early 2020 from an international source, while other counties acted as transmission "sinks." In addition, major policies, such as the end of the lockdown period in 2020 or the end of all restrictions in 2021, did not appear to have an impact on virus diversity across individual counties.

Genomic epidemiology reveals the dominance of Hennepin County in transmission of SARS-CoV-2 in Minnesota from 2020-2022.

SARS-CoV-2 has had an unprecedented impact on human health and highlights the need for genomic epidemiology studies to increase our understanding of virus evolution and spread, and to inform policy decisions. We sequenced viral genomes from over 22,000 patient samples tested at Mayo Clinic Laboratories between 2020-2022 and use Bayesian phylodynamics to describe county and regional spread in Minnesota. The earliest introduction into Minnesota was to Hennepin County from a domestic source around January 22, 2020; six weeks before the first confirmed case in the state. This led to the virus spreading to Northern Minnesota, and eventually, the rest of the state. International introductions were most abundant in Hennepin (home to the Minneapolis/St. Paul International (MSP) airport) totaling 45 (out of 107) over the two-year period. Southern Minnesota counties were most common for domestic introductions with 19 (out of 64), potentially driven by bordering states such as Iowa and Wisconsin as well as Illinois which is nearby. Hennepin also was, by far, the most dominant source of in-state transmissions to other Minnesota locations (n=772) over the two-year period. We also analyzed the diversity of the location source of SARS-CoV-2 viruses in each county and noted the timing of state-wide policies as well as trends in clinical cases. Neither the number of clinical cases or major policy decisions, such as the end of the lockdown period in 2020 or the end of all restrictions in 2021, appeared to have impact on virus diversity across each individual county.

Whole-exome sequencing of transforming oral lichen planus reveals mutations in DNA damage repair and apoptosis pathway genes.

BACKGROUND: Oral lichen planus confers a 1% risk of transformation to oral squamous cell carcinoma. While prior exome sequencing studies have identified multiple genetic mutations in oral squamous cell carcinoma, mutational analyses of lichen planus-derived OSCC are lacking. We sought to clarify genomic events associated with oral lichen planus transformation. METHODS: Using rigorous diagnostic criteria, we retrospectively identified patients with non-transforming oral lichen planus (i.e., known to be non-transforming with 5 years of clinical follow-up; n = 17), transforming oral lichen planus (tissue marginal to oral squamous cell carcinoma, n = 9), or oral squamous cell carcinoma arising in lichen planus (n = 17). Gene mutational profiles derived from whole-exome sequencing on fixed mucosal specimens were compared among the groups. RESULTS: The four most frequently mutated genes in transforming oral lichen planus and oral squamous cell carcinoma (TP53, CELSR1, CASP8, and KMT2D) identified 12/17 (71%) of oral squamous cell carcinomas and 5/9 (56%) of transforming oral lichen planus but were absent in non-transforming oral lichen planus. We identified other known oral squamous cell carcinoma mutations (TRRAP, OBSCN, and LRP2) but also previously unreported mutations (TENM3 and ASH1L) in lichen planus-associated oral squamous cell carcinomas. CONCLUSIONS: These findings suggest alterations in DNA damage response and apoptosis pathways underlie lichen planus-related oral squamous cell carcinoma transformation and are supported by mutational signatures indicative of DNA damage. This study characterized patterns of mutational events present in oral lichen planus associated with squamous cell carcinoma and in squamous cell carcinoma associated with oral lichen planus but not in non-transforming oral lichen planus.

Pathogenesis of pancreatic cancer exosome-induced lipolysis in adipose tissue.

BACKGROUND AND OBJECTIVES: New-onset diabetes and concomitant weight loss occurring several months before the clinical presentation of pancreatic cancer (PC) appear to be paraneoplastic phenomena caused by tumour-secreted products. Our recent findings have shown exosomal adrenomedullin (AM) is important in development of diabetes in PC. Adipose tissue lipolysis might explain early onset weight loss in PC. We hypothesise that lipolysis-inducing cargo is carried in exosomes shed by PC and is responsible for the paraneoplastic effects. Therefore, in this study we investigate if exosomes secreted by PC induce lipolysis in adipocytes and explore the role of AM in PC-exosomes as the mediator of this lipolysis. DESIGN: Exosomes from patient-derived cell lines and from plasma of patients with PC and non-PC controls were isolated and characterised. Differentiated murine (3T3-L1) and human adipocytes were exposed to these exosomes to study lipolysis. Glycerol assay and western blotting were used to study lipolysis. Duolink Assay was used to study AM and adrenomedullin receptor (ADMR) interaction in adipocytes treated with exosomes. RESULTS: In murine and human adipocytes, we found that both AM and PC-exosomes promoted lipolysis, which was abrogated by ADMR blockade. AM interacted with its receptor on the adipocytes, activated p38 and extracellular signal-regulated (ERK1/2) mitogen-activated protein kinases and promoted lipolysis by phosphorylating hormone-sensitive lipase. PKH67-labelled PC-exosomes were readily internalised into adipocytes and involved both caveolin and macropinocytosis as possible mechanisms for endocytosis. CONCLUSIONS: PC-secreted exosomes induce lipolysis in subcutaneous adipose tissue; exosomal AM is a candidate mediator of this effect.

Pancreatic Cancer-Derived Exosomes Cause Paraneoplastic ß-cell Dysfunction.

PURPOSE: Pancreatic cancer frequently causes diabetes. We recently proposed adrenomedullin as a candidate mediator of pancreatic ß-cell dysfunction in pancreatic cancer. How pancreatic cancer-derived adrenomedullin reaches ß cells remote from the cancer to induce ß-cell dysfunction is unknown. We tested a novel hypothesis that pancreatic cancer sheds adrenomedullin-containing exosomes into circulation, which are transported to ß cells and impair insulin secretion. EXPERIMENTAL METHODS: We characterized exosomes from conditioned media of pancreatic cancer cell lines (n = 5) and portal/peripheral venous blood of patients with pancreatic cancer (n = 20). Western blot analysis showed the presence of adrenomedullin in pancreatic cancer-exosomes. We determined the effect of adrenomedullin-containing pancreatic cancer exosomes on insulin secretion from INS-1 ß cells and human islets, and demonstrated the mechanism of exosome internalization into ß cells. We studied the interaction between ß-cell adrenomedullin receptors and adrenomedullin present in pancreatic cancer-exosomes. In addition, the effect of adrenomedullin on endoplasmic reticulum (ER) stress response genes and reactive oxygen/nitrogen species generation in ß cells was shown. RESULTS: Exosomes were found to be the predominant extracellular vesicles secreted by pancreatic cancer into culture media and patient plasma. Pancreatic cancer-exosomes contained adrenomedullin and CA19-9, readily entered ß cells through caveolin-mediated endocytosis or macropinocytosis, and inhibited insulin secretion. Adrenomedullin in pancreatic cancer exosomes interacted with its receptor on ß cells. Adrenomedullin receptor blockade abrogated the inhibitory effect of exosomes on insulin secretion. ß cells exposed to adrenomedullin or pancreatic cancer exosomes showed upregulation of ER stress genes and increased reactive oxygen/nitrogen species. CONCLUSIONS: Pancreatic cancer causes paraneoplastic ß-cell dysfunction by shedding adrenomedullin(+)/CA19-9(+) exosomes into circulation that inhibit insulin secretion, likely through adrenomedullin-induced ER stress and failure of the unfolded protein response.

GAIP interacting protein C-terminus regulates autophagy and exosome biogenesis of pancreatic cancer through metabolic pathways.

GAIP interacting protein C terminus (GIPC) is known to play an important role in a variety of physiological and disease states. In the present study, we have identified a novel role for GIPC as a master regulator of autophagy and the exocytotic pathways in cancer. We show that depletion of GIPC-induced autophagy in pancreatic cancer cells, as evident from the upregulation of the autophagy marker LC3II. We further report that GIPC regulates cellular trafficking pathways by modulating the secretion, biogenesis, and molecular composition of exosomes. We also identified the involvement of GIPC on metabolic stress pathways regulating autophagy and microvesicular shedding, and observed that GIPC status determines the loading of cellular cargo in the exosome. Furthermore, we have shown the overexpression of the drug resistance gene ABCG2 in exosomes from GIPC-depleted pancreatic cancer cells. We also demonstrated that depletion of GIPC from cancer cells sensitized them to gemcitabine treatment, an avenue that can be explored as a potential therapeutic strategy to overcome drug resistance in cancer.

The role of factor inhibiting HIF (FIH-1) in inhibiting HIF-1 transcriptional activity in glioblastoma multiforme.

Glioblastoma multiforme (GBM) accounts for about 38% of primary brain tumors in the United States. GBM is characterized by extensive angiogenesis induced by vascular growth factors and cytokines. The transcription of these growth factors and cytokines is regulated by the Hypoxia-Inducible-Factor-1(HIF-1), which is a key regulator mediating the cellular response to hypoxia. It is known that Factor Inhibiting HIF-1, or FIH-1, is also involved in the cellular response to hypoxia and has the capability to physically interact with HIF-1 and block its transcriptional activity under normoxic conditions. Delineation of the regulatory role of FIH-1 will help us to better understand the molecular mechanism responsible for tumor growth and progression and may lead to the design of new therapies targeting cellular pathways in response to hypoxia. Previous studies have shown that the chromosomal region of 10q24 containing the FIH-1 gene is often deleted in GBM, suggesting a role for the FIH-1 in GBM tumorigenesis and progression. In the current study, we found that FIH-1 is able to inhibit HIF-mediated transcription of GLUT1 and VEGF-A, even under hypoxic conditions in human glioblastoma cells. FIH-1 has been found to be more potent in inhibiting HIF function than PTEN. This observation points to the possibility that deletion of 10q23-24 and loss or decreased expression of FIH-1 gene may lead to a constitutive activation of HIF-1 activity, an alteration of HIF-1 targets such as GLUT-1 and VEGF-A, and may contribute to the survival of cancer cells in hypoxia and the development of hypervascularization observed in GBM. Therefore FIH-1 can be potential therapeutic target for the treatment of GBM patients with poor prognosis.

RGS-GAIP-interacting protein controls breast cancer progression.

Although the importance of RGS-GAIP-interacting protein (GIPC) in the biology of malignant cells is well known, the molecular mechanism of GIPC in the inhibition of tumor progression has not been identified. This study focused on elucidating the molecular role of GIPC in breast cancer progression. By using a human breast tumor specimen, an in vivo mouse model, and breast cancer cell lines, we showed for the first time that GIPC is involved in breast cancer progression through regulation of breast cancer cell proliferation, survival, and invasion. Furthermore, we found that the Akt/Mdm2/p53 axis, insulin-like growth factor-1 receptor, matrix metalloproteinase-9, and Cdc42 were downstream of GIPC signaling in breast cancer cells. Moreover, we showed that wild-type p53 reduced GIPC-induced breast cancer cell survival, whereas mutant p53 inhibited GIPC-induced cell invasion. Finally, we demonstrated that an N-myristoylated GIPC peptide (CR1023, N-myristoyl-PSQSSSEA) capable of blocking the PDZ domain of GIPC successfully inhibited MDA-MB-231 cell proliferation, survival, and further in vivo tumor growth. Taken together, these findings demonstrate the importance of GIPC in breast tumor progression, which has a potentially significant impact on the development of therapies against many common cancers expressing GIPC, including breast and renal cancer.

Insulin receptor substrate-2 mediated insulin-like growth factor-I receptor overexpression in pancreatic adenocarcinoma through protein kinase Cdelta.

Pancreatic adenocarcinoma (PCA) is an almost invariably fatal disease. Recently, it has been shown by several groups as well as ours that insulin-like growth factor-I receptor (IGF-IR) overexpression is related to higher proliferation, survival, angiogenesis, and highly invasive pancreatic tumors. Several studies have been carried out to understand the pathways that lead to growth factor-mediated signaling, but the molecular mechanism of receptor overexpression remains mostly unknown. Treatment with neutralizing antibodies or a specific kinase inhibitor against IGF-IR could block the receptor expression in PCA cells. Furthermore, we also showed that insulin receptor substrate (IRS)-2, but not IRS-1, is involved in regulation of IGF-IR expression, which is most likely not transcriptional control. By blocking mammalian target of rapamycin (mTOR) pathway with rapamycin as well as other biochemical analysis, we defined a unique regulation of IGF-IR expression mediated by protein kinase Cdelta (PKCdelta) and mTOR pathway. Moreover, we showed that the down-regulation of IGF-IR expression due to IRS-2 small interfering RNA can be compensated by overexpression of dominant-active mutant of PKCdelta, suggesting that PKCdelta is downstream of IGF-IR/IRS-2 axis. Overall, these findings suggest a novel regulatory role of IRS-2 on the expression of IGF-IR through PKCdelta and mTOR in pancreatic cancer cells.

Targeted delivery of gemcitabine to pancreatic adenocarcinoma using cetuximab as a targeting agent.

One of the key challenges in anticancer therapy is the toxicity and poor bioavailability of the anticancer drugs. Nanotechnology can play a pivotal role by delivering drugs in a targeted fashion to the malignant cells that will reduce the systemic toxicity of the anticancer drug. In this report, we show a stepwise development of a nanoparticle-based targeted delivery system for in vitro and in vivo therapeutic application in pancreatic cancer. In the first part of the study, we have shown the fabrication and characterization of the delivery system containing gold nanoparticle as a delivery vehicle, cetuximab as a targeting agent, and gemcitabine as an anticancer drug for in vitro application. Nanoconjugate was first characterized physico-chemically. In vitro targeting efficacy, tested against three pancreatic cancer cell lines (PANC-1, AsPC-1, and MIA Paca2) with variable epidermal growth factor receptor (EGFR) expression, showed that gold uptake correlated with EGFR expression. In the second part, we showed the in vivo therapeutic efficacy of the targeted delivery system. Administration of this targeted delivery system resulted in significant inhibition of pancreatic tumor cell proliferation in vitro and orthotopic pancreatic tumor growth in vivo. Tumor progression was monitored noninvasively by measuring bioluminescence of the implanted tumor cells. Pharmacokinetic experiments along with the quantitation of gold both in vitro and in vivo further confirmed that the inhibition of tumor growth was due to targeted delivery. This strategy could be used as a generalized approach for the treatment of a variety of cancers characterized by overexpression of EGFR.

Protein kinase C-mediated modulation of FIH-1 expression by the homeodomain protein CDP/Cut/Cux.

Under normoxia, FIH-1 (factor inhibiting HIF-1) inhibits the transcriptional activity of hypoxia-inducible factor (HIF); however, under such conditions, we observed a significant level of HIF activity in renal cell carcinoma (RCC). This phenomenon could be attributed to a decrease in the level of functional FIH that has been identified in our previous work. Nonetheless, the molecular mechanism of FIH regulation in cancer, in particular RCC, was unclear until now. In this communication, we have demonstrated that in RCC, the Cut-like homeodomain protein (CDP/Cut) is involved in FIH transcriptional regulation and is controlled by a specific signaling event involving protein kinase C (PKC) zeta. Furthermore, we have defined a unique CDP/Cut binding site on the FIH promoter. With chromatin immunoprecipitation assays, we show that CDP binds to the FIH-1 promoter in vivo and that this binding is PKC zeta dependent. Moreover, we have also defined a potential phosphorylation site in CDP (serine 987) that modulates FIH expression. CDP/Cut is a transcriptional repressor that decreases FIH-1 expression and subsequently leads to a decrease in the repressor activity of FIH-1. Without this repression, HIF activity increases, allowing for the increased transcription of the genes it regulates, such as the vascular endothelial growth factor and GLUT-1 genes. Both CDP and HIF levels are increased in several cancers and are responsible for the metastatic progression of the tumors. Taken together, our results suggest for the first time a potential connection between CDP and FIH that could lead to the development of future therapeutic interventions.

Expression and regulatory role of GAIP-interacting protein GIPC in pancreatic adenocarcinoma.

Regulator of G-protein signaling-GAIP-interacting protein COOH terminus (GIPC) is involved in protein trafficking, endocytosis, and receptor clustering and is associated with insulin-like growth factor I receptor (IGF-IR), a receptor important for proliferation and anchorage-independent growth. Here, we described GIPC expression in different human pancreatic adenocarcinoma (PCA) cell lines and we examined the role of GIPC in the regulation of IGF-IR protein levels in PCA. Interestingly, inhibition of GIPC expression by RNA interference led to reduced IGF-IR protein levels and a subsequent decrease in proliferation of PCA cells. We also determined that the PDZ domain of GIPC is essential for the post-translational regulation and the binding of IGF-IR. The importance of GIPC in pancreatic cancer development and progression is supported by tissue microarray data of 300 pancreatic cancer specimens where GIPC is highly expressed in PCA. Taken together, our data suggest that GIPC is a central molecule for the stability of IGF-IR and could be a target for future therapy.

Cigarette smoke extract suppresses human dendritic cell function leading to preferential induction of Th-2 priming.

Dendritic cells (DC) are key regulators of immune responses. In the current study, we hypothesized that cigarette smoke-induced aberrance in DC function is an important mechanism by which smokers develop cancer, infection, and allergy--diseases common in smokers. We demonstrate that cigarette smoke extract (CSE) inhibits DC-mediated priming of T cells, specifically inhibiting the secretion of IFN-gamma whereas enhancing the production of IL-4 in the MLR. Conditioning with CSE did not effect cytokine (IL-10, IL-6, or IL-12) production from immature DCs, but significantly inhibited IL-12p70 release by LPS-matured DCs. In contrast, IL-10 secretion by LPS-activated CSE-conditioned DCs was enhanced when compared with control DCs. CSE also induced cyclooxygenase-2 protein levels in maturing DCs and significantly augmented endogenous PGE2 release. Conditioning of DCs with CSE also suppressed LPS-mediated induction of CD40, CD80, and CD86, and suppressed maturation-associated CCR7 expression. Although CSE has been reported to induce apoptosis of fibroblasts and epithelial cells, the immunomodulatory effects observed with CSE were not due to diminished DC viability. The effects of CSE on DC function were not exclusively mediated by nicotine, because equivalent, or even higher concentrations of nicotine than those found in CSE, failed to suppress DC-induced T cell priming. These data provide evidence that soluble components extracted from cigarette smoke suppress key DC functions and favor the development of Th-2 immunity.

Vascular endothelial growth factor impairs the functional ability of dendritic cells through Id pathways.

Vascular endothelial growth factor (VEGF) is an angiogenic cytokine that plays an important role in tumor growth and progression. Recent evidence suggests an alternate, albeit indirect, role of VEGF on host immune response to tumors. VEGF appears to diminish host immunity by altering the function of major antigen-presenting cells such as dendritic cells (DCs) [D.I. Gabrilovich, T. Ishida, S. Nadaf, J.E. Ohm, D.P. Carbone, Antibodies to vascular endothelial growth factor enhance the efficacy of cancer immunotherapy by improving endogenous dendritic cell function, Clin. Cancer Res. 5 (1999) 2963-2970, D. Gabrilovich, T. Ishida, T. Oyama, S. Ran, V. Kravtsov, S. Nadaf, D.P. Carbone, Vascular endothelial growth factor inhibits the development of dendritic cells and dramatically affects the differentiation of multiple hematopoietic lineages in vivo, Blood 92 (1998) 4150-4166, T. Oyama, S. Ran, T. Ishida, S. Nadaf, L. Kerr, D.P. Carbone, D.I. Gabrilovich, Vascular endothelial growth factor affects dendritic cell maturation through the inhibition of nuclear factor-kappa B activation in hemopoietic progenitor cells, J. Immunol. 160 (1998) 1224-1232.]. DCs are prime initiators of host immunity as they are known to activate both primary as well as secondary immune responses [J. Banchereau, F. Briere, C. Caux, J. Davoust, S. Lebecque, Y.J. Liu, B. Pulendran, K. Palucka, Immunobiology of dendritic cells, Ann. Rev. Immunol. 18 (2000) 767-811.]. However, the exact nature of how VEGF suppresses DC function is not fully clear. In this report, we show that DCs cultured in the presence of VEGF are less potent in stimulating antigen-specific T-cells. Furthermore, by using DCs derived from Id1(-/-) mice that are defective in Flt-1 signaling, we demonstrated that the inhibitory function of VEGF on DC function is most likely mediated by Flt-1. Thus, the role of VEGF in downregulating host immunity may highlight a unique role of VEGF in the pathogenesis of cancer.

Blockade of B7-H1 and PD-1 by monoclonal antibodies potentiates cancer therapeutic immunity.

Contemporary approaches for vaccination and immunotherapy are often capable of eliciting strong T-cell responses against tumor antigens. However, such responses are not parallel to clinical tumor regression. The development of evasion mechanisms within tumor microenvironment may be responsible for poor therapeutic responses. We report here that constitutive or inducible expression of B7-H1, a B7 family molecule widely expressed by cancers, confers resistance to therapeutic anti-CD137 antibody in mice with established tumors. The resistance is accompanied with failure of antigen-specific CD8+ CTLs to destroy tumor cells without impairment of CTL function. Blockade of B7-H1 or PD-1 by specific monoclonal antibodies could reverse this resistance and profoundly enhance therapeutic efficacy. Our findings support that B7-H1/PD-1 forms a molecular shield to prevent destruction by CTLs and implicate new approaches for immunotherapy of human cancers.

B7-H3 enhances tumor immunity in vivo by costimulating rapid clonal expansion of antigen-specific CD8+ cytolytic T cells.

B7-H3 is a B7 family molecule with T cell costimulatory function in vitro. The in vivo role of B7-H3 in the stimulation of tumor immunity is unclear. We report here that expression of B7-H3 by transfection of the mouse P815 tumor line enhances its immunogenicity, leading to the regression of tumors and amplification of a tumor-specific CD8+ CTL response in syngeneic mice. Tumor cells engineered to express B7-H3 elicit a rapid clonal expansion of P1A tumor Ag-specific CD8+ CTL in lymphoid organs in vivo and acquire the ability to directly stimulate T cell growth, division, and development of cytolytic activity in vitro. Our results thus establish a role for B7-H3 in the costimulation of T cell immune responses in vivo.

53BP1 is required for class switch recombination.

53BP1 participates early in the DNA damage response and is involved in cell cycle checkpoint control. Moreover, the phenotype of mice and cells deficient in 53BP1 suggests a defect in DNA repair (Ward et al., 2003b). Therefore, we asked whether or not 53BP1 would be required for the efficient repair of DNA double strand breaks. Our data indicate that homologous recombination by gene conversion does not depend on 53BP1. Moreover, 53BP1-deficient mice support normal V(D)J recombination, indicating that 53BP1 is not required for "classic" nonhomologous end joining. However, class switch recombination is severely impaired in the absence of 53BP1, suggesting that 53BP1 facilitates DNA end joining in a way that is not required or redundant for the efficient closing of RAG-induced strand breaks. These findings are similar to those observed in mice or cells deficient in the tumor suppressors ATM and H2AX, further suggesting that the functions of ATM, H2AX, and 53BP1 are closely linked.

Genomic organization and expression analysis of B7-H4, an immune inhibitory molecule of the B7 family.

B7-H4 is a recently identified B7 family member that negatively regulates T cell immunity by the inhibition of T cell proliferation, cytokine production, and cell cycle progression. In this study, we report that the genomic DNA of human B7-H4 is mapped on chromosome 1 comprised of six exons and five introns spanning 66 kb, of which exon 6 is used for alternative splicing to generate two different transcripts. Similar B7-H4 structure is also found in mouse genomic DNA in chromosome 3. A human B7-H4 pseudogene is identified in chromosome 20p11.1 with a single exon and two stop codons in the coding region. Immunohistochemistry analysis using B7-H4-specific mAb demonstrates that B7-H4 is not expressed on the majority of normal human tissues. In contrast, up to 85% (22 of 26) of ovarian cancer and 31% (5 of 16) of lung cancer tissues constitutively express B7-H4. Our results indicate a tight regulation of B7-H4 expression in the translational level in normal peripheral tissues and a potential role of B7-H4 in the evasion of tumor immunity.