Radiation modulates the peptide repertoire, enhances MHC class I expression, and induces successful antitumor immunotherapy.

Radiotherapy is one of the most successful cancer therapies. Here the effect of irradiation on antigen presentation by MHC class I molecules was studied. Cell surface expression of MHC class I molecules was increased for many days in a radiation dose-dependent manner as a consequence of three responses. Initially, enhanced degradation of existing proteins occurred which resulted in an increased intracellular peptide pool. Subsequently, enhanced translation due to activation of the mammalian target of rapamycin pathway resulted in increased peptide production, antigen presentation, as well as cytotoxic T lymphocyte recognition of irradiated cells. In addition, novel proteins were made in response to gamma-irradiation, resulting in new peptides presented by MHC class I molecules, which were recognized by cytotoxic T cells. We show that immunotherapy is successful in eradicating a murine colon adenocarcinoma only when preceded by radiotherapy of the tumor tissue. Our findings indicate that directed radiotherapy can improve the efficacy of tumor immunotherapy.

The breast cancer resistance protein BCRP (ABCG2) concentrates drugs and carcinogenic xenotoxins into milk.

Contamination of milk with drugs, pesticides and other xenotoxins can pose a major health risk to breast-fed infants and dairy consumers. Here we show that the multidrug transporter BCRP (encoded by ABCG2) is strongly induced in the mammary gland of mice, cows and humans during lactation and that it is responsible for the active secretion of clinically and toxicologically important substrates such as the dietary carcinogen PhIP, the anticancer drug topotecan and the antiulcerative cimetidine into mouse milk.

TCR gene therapy of spontaneous prostate carcinoma requires in vivo T cell activation.

Analogous to the clinical use of recombinant high-affinity Abs, transfer of TCR genes may be used to create a T cell compartment specific for self-Ags to which the endogenous T cell repertoire is immune tolerant. In this study, we show in a spontaneous prostate carcinoma model that the combination of vaccination with adoptive transfer of small numbers of T cells that are genetically modified with a tumor-specific TCR results in a marked suppression of tumor development, even though both treatments are by themselves without effect. These results demonstrate the value of TCR gene transfer to target otherwise nonimmunogenic tumor-associated self-Ags provided that adoptive transfer occurs under conditions that allow in vivo expansion of the TCR-modified T cells.

Methotrexate pharmacokinetics in transgenic mice with liver-specific expression of human organic anion-transporting polypeptide 1B1 (SLCO1B1).

Human organic anion-transporting polypeptide 1B1 (OATP1B1) is an important hepatic uptake transporter that can transport a wide variety of drugs. In the present study, we have generated and characterized a transgenic mouse model with specific and functional expression of human OATP1B1 (SLCO1B1) in the liver. Immunohistochemical staining revealed basolateral localization of transgenic OATP1B1 in the liver, whereas no expression of OATP1B1 was found in the kidney and small intestine. Using this transgenic model, the in vivo role of human OATP1B1 in the disposition of the anticancer drug methotrexate (MTX) was studied. In mice on a semisynthetic diet, the area under the plasma concentration-time curve for intravenous methotrexate in SLCO1B1 transgenic mice was 1.5-fold decreased compared with wild-type mice. Furthermore, the amount of MTX in the liver was markedly higher ( approximately 2-fold) in the SLCO1B1 transgenic mice compared with wild-type mice, resulting in 2- to 4-fold higher liver-plasma ratios of MTX. Some murine liver Slco genes were markedly down-regulated on the semisynthetic diet compared with a standard diet, which probably reduced murine Oatp-mediated MTX uptake in the liver and therefore facilitated detection of the function of the transgenic OATP1B1. Taken together, these data demonstrate a marked and possibly rate-limiting role for human OATP1B1 in MTX elimination in vivo. Variation in OATP1B1 activity due to genetic polymorphisms, drug-drug interactions, and possibly dietary conditions may therefore play a role in the severity of MTX-related toxicity. SLCO1B1 transgenic mice could be a useful tool in studying the in vivo role of human OATP1B1 in drug pharmacokinetics.

Radiosensitization of squamous cell carcinoma by the alkylphospholipid perifosine in cell culture and xenografts.

PURPOSE: Combined modality treatment has improved outcome in various solid tumors. Besides classic anticancer drugs, a new generation of biological response modifiers has emerged that increases the efficacy of radiation. Here, we have investigated whether perifosine, an orally applicable, membrane-targeted alkylphospholipid, enhances the antitumor effect of radiation in vitro and in vivo. EXPERIMENTAL DESIGN: Several long-term and short-term in vitro assays (clonogenic survival, sulforhodamine B cytotoxicity, apoptosis, and cell cycle analysis) were used to assess the cytotoxic effect of perifosine in combination with radiation. In vivo, the response of human KB squamous cell carcinoma xenografts was measured after treatment with perifosine, irradiation, and the combination. Radiolabeled perifosine was used to determine drug disposition in tumor and normal tissues. At various intervals after treatment, tumor specimens were collected to document histopathologic changes. RESULTS: In vitro, perifosine reduced clonogenic survival, enhanced apoptosis, and increased cell cycle arrest after radiation. In vivo, radiation and perifosine alone induced a dose-dependent tumor growth delay. When combining multiple perifosine administrations with single or split doses of radiation, complete and sustained tumor regression was observed. Histopathologic analysis of tumor specimens revealed a prominent apoptotic response after combined treatment with radiation and perifosine. Radiation-enhanced tumor response was observed at clinically relevant plasma perifosine concentrations and accumulating drug disposition of >100 microg/g in tumor tissue. CONCLUSIONS: Perifosine enhances radiation-induced cytotoxicity, as evidenced by reduced clonogenic survival and increased apoptosis induction in vitro and by complete tumor regression in vivo. These data provide strong support for further development of this combination in clinical studies.

Mammary-specific inactivation of E-cadherin and p53 impairs functional gland development and leads to pleomorphic invasive lobular carcinoma in mice.

Breast cancer is the most common malignancy in women of the Western world. Even though a large percentage of breast cancer patients show pathological complete remission after standard treatment regimes, approximately 30-40% are non-responsive and ultimately develop metastatic disease. To generate a good preclinical model of invasive breast cancer, we have taken a tissue-specific approach to somatically inactivate p53 and E-cadherin, the cardinal cell-cell adhesion receptor that is strongly associated with tumor invasiveness. In breast cancer, E-cadherin is found mutated or otherwise functionally silenced in invasive lobular carcinoma (ILC), which accounts for 10-15% of all breast cancers. We show that mammary-specific stochastic inactivation of conditional E-cadherin and p53 results in impaired mammary gland function during pregnancy through the induction of anoikis resistance of mammary epithelium, resulting in loss of epithelial organization and a dysfunctional mammary gland. Moreover, combined inactivation of E-cadherin and p53 induced lactation-independent development of invasive and metastatic mammary carcinomas, which showed strong resemblance to human pleomorphic ILC. Dissemination patterns of mouse ILC mimic the human malignancy, showing metastasis to the gastrointestinal tract, peritoneum, lung, lymph nodes and bone. Our results confirm that loss of E-cadherin contributes to both mammary tumor initiation and metastasis, and establish a preclinical mouse model of human ILC that can be used for the development of novel intervention strategies to treat invasive breast cancer.

Regulation of the p27(Kip1) tumor suppressor by miR-221 and miR-222 promotes cancer cell proliferation.

MicroRNAs (miRNAs) are potent post-transcriptional regulators of protein coding genes. Patterns of misexpression of miRNAs in cancer suggest key functions of miRNAs in tumorigenesis. However, current bioinformatics tools do not entirely support the identification and characterization of the mode of action of such miRNAs. Here, we used a novel functional genetic approach and identified miR-221 and miR-222 (miR-221&222) as potent regulators of p27(Kip1), a cell cycle inhibitor and tumor suppressor. Using miRNA inhibitors, we demonstrate that certain cancer cell lines require high activity of miR-221&222 to maintain low p27(Kip1) levels and continuous proliferation. Interestingly, high levels of miR-221&222 appear in glioblastomas and correlate with low levels of p27(Kip1) protein. Thus, deregulated expression of miR-221&222 promotes cancerous growth by inhibiting the expression of p27(Kip1).

Targeting self-antigens through allogeneic TCR gene transfer.

Adoptive transfer of T-cell receptor (TCR) genes has been proposed as an attractive approach for immunotherapy in cases where the endogenous T-cell repertoire is insufficient. While there are promising data demonstrating the capacity of TCR-modified T cells to react to foreign antigen encounter, the feasibility of targeting tumor-associated self-antigens has not been addressed. Here we demonstrate that T-cell receptor gene transfer allows the induction of defined self-antigen-specific T-cell responses, even when the endogenous T-cell repertoire is nonreactive. Furthermore, we show that adoptive transfer of T-cell receptor genes can be used to induce strong antigen-specific T-cell responsiveness in partially MHC-mismatched hosts without detectable graft versus host disease. These results demonstrate the feasibility of using a collection of "off the shelf" T-cell receptor genes to target defined tumor-associated self-antigens and thereby form a clear incentive to test this immunotherapeutic approach in a clinical setting.