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ErbB2/Neu oncogene is overexpressed in 25% of invasive/metastatic breast cancers. We have found that deletion of heat shock factor Hsf1 in mice overexpressing ErbB2/Neu significantly reduces mammary tumorigenesis and metastasis. Hsf1(+/-)ErbB2/Neu(+) tumors exhibit reduced cellular proliferative and invasive properties associated with reduced activated ERK1/2 and reduced epithelial-mesenchymal transition (EMT). Hsf1(+/+)Neu(+) mammary epithelial cells exposed to TGFβ show high levels of ERK1/2 activity and EMT; this is associated with reduced expression of E-cadherin and increased expression of Slug and vimentin, a mesenchymal marker. In contrast, Hsf1(-/-)Neu(+) or Hsf1(+/+)Neu(+) cells do not exhibit activated ERK1/2 and show reduced EMT in the presence of TGFβ. The ineffective activation of the RAS/RAF/MEK/ERK1/2 signaling pathway in cells with reduced levels of HSF1 is due to the low levels of HSP90 in complex with RAF1 that are required for RAF1 stability and maturation. These results indicate a powerful inhibitory effect conferred by HSF1 downstream target genes in the inhibition of ErbB2-induced breast cancers in the absence of the Hsf1 gene.

Summary The inhibitory effects of cancer on T cell metabolism have been well established, but the metabolic impact of immunotherapy on tumor cells is poorly understood. Here, we developed a CD4+ T cell-based adoptive immunotherapy protocol that was curative for mice with implanted colorectal tumors. By conducting metabolic profiling on tumors, we show that adoptive immunotherapy profoundly altered tumor metabolism, resulting in glutathione depletion and accumulation of reactive oxygen species (ROS) in tumor cells. We further demonstrate that T cell-derived tumor necrosis factor alpha (TNF-α) can synergize with chemotherapy to intensify oxidative stress and tumor cell death in an NADPH (nicotinamide adenine dinucleotide phosphate hydrogen) oxidase-dependent manner. Reduction of oxidative stress, by preventing TNF-α-signaling in tumor cells or scavenging ROS, antagonized the therapeutic effects of adoptive immunotherapy. Conversely, provision of pro-oxidants after chemotherapy can partially recapitulate the antitumor effects of T cell transfer. These findings imply that reinforcing tumor oxidative stress represents an important mechanism underlying the efficacy of adoptive immunotherapy.

Immunotherapy of rheumatoid arthritis (RA) using oral-dosed native chicken or bovine type II collagen (nCII) to induce specific immune tolerance is an attractive strategy. However, the majority of clinical trials of oral tolerance in human diseases including RA in recent years have been disappointing. Here, we describe a novel recombinant peptide rcCTE1-2 which contains only two tolerogenic epitopes (CTE1 and CTE2) of chicken type II collagen (cCII). These are the critical T-cell determinants for suppression of RA that were first developed and used to compare its suppressive effects with ncCII on the collagen-induced arthritis (CIA) model. The rcCTE1-2 was produced using the prokaryotic pET expression system and purified by Ni-NTA His affinity chromatography. Strikingly, our results showed clearly that rcCTE1-2 was as efficacious as ncCII at the dose of 50 microg/kg/d. This dose significantly reduced footpad swelling, arthritic incidence and scores, and deferred the onset of disease. Furthermore, rcCTE1-2 of 50 microg/kg/d could lower the level of anti-nCII antibody in the serum of CIA animals, decrease Th1-cytokine INF-gamma level, and increase Th3-cytokine TGF-beta(1) produced level by spleen cells from CIA mice after in vivo stimulation with ncCII. Importantly, rcCTE1-2 was even more potent than native cCII, which was used in the clinic for RA. Equally importantly, the findings that the major T-cell determinants of cCII that are also recognized by H-2(b) MHC-restricted T cells have not previously been reported. Taken together, these results suggest that we have successfully developed a novel recombinant peptide rcCTE1-2 that can induce a potent tolerogenic response in CIA.

Chicken type II procollagen (ccol2a1) has become as an important oral tolerance protein for effective treatment of rheumatoid arthritis. However, its molecular identity remains unclear. Here, we reported the full-length cDNA and nearly complete genomic DNA encoding ccol2a1. We have determined the structural organization, evolutional characters, developmental expression and chromosomal mapping of the gene. The full-length cDNA sequence spans 4837 bp containing all the coding region of the ccol2a1 including 3' and 5' untranslation region. The deduced peptide of ccol2a1, composed of 1420 amino acids, can be divided into signal peptide, N-propeptide, N-telopeptide, triple helix, C-telopeptide and C-propeptide. The ccol2a1 genomic DNA sequence was determined to be 12,523 bp long containing 54 exons interrupted by 53 introns. Comparison of the ccol2a1 with its counterparts in human, mouse, canine, horse, rat, frog and newt revealed highly conserved sequence in the triple helix domain. Chromosomal mapping of ccol2a1 locates it on 4P2. While the ccol2a1 mRNA was expressed in multiple tissues, the protein was only detected in chondrogenic cartilage, vitreous body and cornea. The ccol2a1 was found to contain two isoforms detected by RT-PCR. The distribution of the ccol2a1 lacking exon 2wasfrequently detected in chondrogenic tissues, whereas the exon 2-containing isoform was more abundant in non-chondrogenic tissues. These results provide useful information for preparing recombinant chicken type II collagen and for a better understanding of normal cartilage development.