Doctor of Philosophy, Baylor College Of Medicine (2011)
Monte Winslow, Postdoctoral Faculty Sponsor
Pancreatic ductal adenocarcinoma (PDAC) is a genomically diverse, prevalent, and almost invariably fatal malignancy. Although conventional genetically engineered mouse models of human PDAC have been instrumental in understanding pancreatic cancer development, these models are much too labor-intensive, expensive, and slow to perform the extensive molecular analyses needed to adequately understand this disease. Here we demonstrate that retrograde pancreatic ductal injection of either adenoviral-Cre or lentiviral-Cre vectors allows titratable initiation of pancreatic neoplasias that progress into invasive and metastatic PDAC. To enable in vivo CRISPR/Cas9-mediated gene inactivation in the pancreas, we generated a Cre-regulated Cas9 allele and lentiviral vectors that express Cre and a single-guide RNA. CRISPR-mediated targeting of Lkb1 in combination with oncogenic Kras expression led to selection for inactivating genomic alterations, absence of Lkb1 protein, and rapid tumor growth that phenocopied Cre-mediated genetic deletion of Lkb1. This method will transform our ability to rapidly interrogate gene function during the development of this recalcitrant cancer.
View details for DOI 10.1101/gad.264861.115
View details for Web of Science ID 000358596300010
Despite its clinical importance, very little is known about the natural history and molecular underpinnings of lung cancer dissemination and metastasis. Here we employed a genetically-engineered mouse model of metastatic lung adenocarcinoma in which cancer cells are fluorescently marked to determine whether dissemination is an inherent ability or a major acquired phenotype during lung adenocarcinoma metastasis. We find very little evidence for dissemination from oncogenic Kras-driven hyperplasias or most adenocarcinomas. p53 loss is insufficient to drive dissemination but rather enables rare cancer cells in a small fraction of primary adenocarcinomas to gain alterations that drive dissemination. Molecular characterization of disseminated tumors cells indicates that down-regulation of the transcription factor Nkx2-1 precedes dissemination. Finally, we show that metastatic primary tumors possess a highly proliferative sub-population of cells with characteristics matching those of disseminating cells. We propose that dissemination is a major hurdle during the natural course of lung adenocarcinoma metastasis.
View details for DOI 10.1158/2159-8290.CD-13-0862
View details for Web of Science ID 000338708900024
The activation of innate and adaptive immunity is always balanced by inhibitory signalling mechanisms to maintain tissue integrity. We have identified the E3 ligase c-Cbl--known for its roles in regulating lymphocyte signalling--as a modulator of dendritic cell activation. In c-Cbl-deficient dendritic cells, Toll-like receptor-induced expression of proinflammatory factors, such as interleukin-12, is increased, correlating with a greater potency of dendritic-cell-based vaccines against established tumours. This proinflammatory phenotype is accompanied by an increase in nuclear factor (NF)-?B activity. In addition, c-Cbl deficiency reduces both p50 and p105 levels, which have been shown to modulate the stimulatory function of NF-?B. Our data indicate that c-Cbl has a crucial, RING-domain-dependent role in regulating dendritic cell maturation, probably by facilitating the regulatory function of p105 and/or p50.
View details for DOI 10.1038/embor.2011.143
View details for Web of Science ID 000294476500021
View details for PubMedID 21799517
Tumor-infiltrating lymphocytes (TILs) develop as manifestations of the recognition and defense against malignant cells by the host immune system. TILs were literally defined as "tumor-infiltrating lymphocytes", which a posteriori locate within the tumor tissues. Although such cells can be found, they fail to control the growth of tumor. Many have proposed diverse mechanisms for dysfunction of TILs with regard to the roles of immunosurveillance against cancer. However, only a few cancer types, e.g. melanoma, have seen the benefits brought by activating these cells for immunotherapy. Functional defects of TILs have been linked to abnormalities of signaling molecules; however, there is conflicting data. The death of TILs was attributed to expression of cancer-derived FasL, PD-1 and RCAS1, and cancer-induced activation-induced cell death (AICD). Confirmed by studies using TILs and animal models, the compromise of tumor-specific immune responses was thought to result from not only mechanisms of clonal anergy but also exhaustion and/or deletion. Furthermore, functional cytotoxic CD8(+) TILs might be rendered incompetent by cancer-induced up-regulation of inhibitory NK receptors or proximal signaling abnormalities. Additionally, immune privilege was partly attributed to recruitment of regulatory T cells to the tumor sites. The failure of IL-2 signaling, which stands at the center of T cell functionalities, had been linked to the enzymatic activity of cancer-derived matrix metalloproteinases (MMPs). Finally, the exploitation of IDO expression, an important enzyme in pregnancy-related immunosuppression, by cancer cells might play a role in tumor immunity. The disparity of cancer types, origin, developmental stages and individual genetic backgrounds likely account for differences, or even contradictions, which might be the reason why immunotherapy works only on a few cancer types. Delineating the mechanisms behind functional defects of TILs can help not only boost chances of the development of a successful cure but understand the not fully identified roles played by immune system in the face of malignancies.
View details for DOI 10.1016/j.jri.2005.06.002
View details for Web of Science ID 000232933000004
View details for PubMedID 16111767
In human cervical cancer (CC), local immunity against this human papilloma virus (HPV)-associated neoplasia has been signified. To stratify the possibility of HPV integration on HLA mutations, we measured the genotypic and phenotypic integrity of all available HLA class I loci in 30 cases of CC. Paired normal and cancer genomic DNA was analyzed with DNA typing trays, including 57 subtypes of HLA-A, 120 subtypes of HLA-B, and 60 subtypes of HLA-C. We demonstrated significant mutations of HLA genotype with reduced HLA molecule expression in CC. HPV coincide in > 70% cases of aberrant HLA genes. Southern blot analysis revealed the presence of HPV DNA within the mutated HLA foci. Our study reveals a plausible role of HPV integration in the contexts of aberrant HLA genotypes in CC cells. Disruptions of the HLA genes can be possible tactics of HPV to attain the potential carcinogenetic purposes, and thus the cancer immune escape.
View details for DOI 10.1016/j.clim.2005.02.001
View details for Web of Science ID 000229390300009
View details for PubMedID 15893697
Inhibitory signals that govern the cytolytic functions of CD8(+) T lymphocytes have been linked to the expression of natural killer cell receptors (NKRs) on CTLs. There is limited knowledge about the induction of inhibitory NKR (iNKR) expression in vivo. Up-regulation of iNKRs has been linked to the modulation of the virus- and/or tumor-specific immune responses in animal models. In the present study, we directly examined the expression of various NKRs on tumor-infiltrating lymphocytes (TILs) derived from human cervical cancer. We found that in human cervical cancer, the percentage expression of immunoglobulin-like NKR(+)CD8(+) T lymphocytes were similar in gated CD8(+)-autologous TILs and peripheral blood mononuclear cells. On the contrary, cervical cancer-infiltrating CD8(+) T lymphocytes expressed up-regulated C-type lectin NKRs CD94/NKG2A compared with either peripheral blood CD8(+) T cells or normal cervix-infiltrating CD8(+) T lymphocytes. Dual NKR coexpression analyses showed that CD94 and NKG2A were mainly expressed on CD56(-)CD161(-)CD8(+) TILs within the cancer milieu. Immunohistochemical study showed that cervical cancer cells expressed abundant interleukin 15 (IL-15) and transforming growth factor-beta (TGF-beta). In kinetic coculture assay, cervical cancer cells can promote the expression of CD94/NKG2A on CD8(+) T lymphocytes. The cancer-derived effects can be reversed by addition of rIL-15Ralpha/Fc and anti-TGF-beta antibody. Functional analyses illustrated that intracellular perforin expression of CD8(+) T cells was minimal upon up-regulation of CD94/NKG2A. Kinetic cytotoxicity assays showed that up-regulated expressions of CD94/NKG2A restrain CD8(+) T lymphocyte cytotoxicity. Our study strongly indicated that cervical cancer cells could promote the expression of iNKRs via an IL-15- and possibly TGF-beta-mediated mechanism and abrogate the antitumor cytotoxicity of TILs.
View details for Web of Science ID 000227972300057
View details for PubMedID 15805295