Erlotinib and bevacizumab in patients with recurrent or metastatic squamous-cell carcinoma of the head and neck: a phase I/II study.

BACKGROUND: Epidermal growth factor receptor (EGFR) is a validated target in squamous-cell carcinoma of the head and neck, but in patients with recurrent or metastatic disease, EGFR targeting agents have displayed modest efficacy. Vascular endothelial growth factor (VEGF)-mediated angiogenesis has been implicated as a mechanism of resistance to anti-EGFR therapy. In this multi-institutional phase I/II study we combined an EGFR inhibitor, erlotinib, with an anti-VEGF antibody, bevacizumab. METHODS: Between April 15, 2003, and Jan 27, 2005, patients with recurrent or metastatic squamous-cell carcinoma of the head and neck were enrolled from seven centres in the USA and were given erlotinib (150 mg daily) and bevacizumab in escalating dose cohorts. The primary objectives in the phase I and II sections, respectively, were to establish the maximum tolerated dose and dose-limiting toxicity of bevacizumab when administered with erlotinib and to establish the proportion of objective responses and time to disease progression. Pretreatment serum and tissues were collected and analysed by enzyme-linked immunosorbent assay and immunofluorescence quantitative laser analysis, respectively. This study was registered with ClinicalTrials.gov, number NCT00055913. FINDINGS: In the phase I section of the trial, ten patients were enrolled in three successive cohorts with no dose-limiting toxic effects noted. 46 patients were enrolled in the phase II section of the trial (including three patients from the phase I section) on the highest dose of bevacizumab (15 mg/kg every 3 weeks). Two additional patients were accrued beyond the protocol-stipulated 46, leaving a total of 48 patients for the phase II assessment. The most common toxic effects of any grade were rash and diarrhoea (41 and 16 of 48 patients, respectively). Three patients had serious bleeding events of grade 3 or higher. Seven patients had a response, with four showing a complete response allowing rejection of the null hypothesis. Median time of overall survival and progression-free survival (PFS) were 7.1 months (95% CI 5.7-9.0) and 4.1 months (2.8-4.4), respectively. Higher ratios of tumour-cell phosphorylated VEGF receptor-2 (pVEGFR2) over total VEGFR2 and endothelial-cell pEGFR over total EGFR in pretreatment biopsies were associated with complete response (0.704 vs 0.386, p=0.036 and 0.949 vs 0.332, p=0.036, respectively) and tumour shrinkage (p=0.007 and p=0.008, respectively) in a subset of 11 patients with available tissue. INTERPRETATION: The combination of erlotinib and bevacizumab is well tolerated in recurrent or metastatic squamous-cell carcinoma of the head and neck. A few patients seem to derive a sustained benefit and complete responses were associated with expression of putative targets in pretreatment tumour tissue.

Interferon-inducible protein IFIXalpha inhibits cell invasion by upregulating the metastasis suppressor maspin.

IFIXalpha, a member of the interferon-inducible HIN-200 family, has been identified as a putative tumor suppressor. However, the molecular mechanisms underlying IFIXalpha-mediated tumor suppression are poorly understood. In the present study, we demonstrated that the metastasis suppressor maspin acts as the downstream target of IFIXalpha. IFIXalpha suppressed the invasion activity of MDA-MB-468 breast cancer cells, and its inhibitory effect was reversed by the knockdown of maspin. Both Maspin mRNA and protein were upregulated by IFIXalpha. Histone deacetylase (HDAC) inhibitors, but not DNA methyltransferase inhibitor upregulated maspin, and HDAC1 inhibited the transactivation of maspin promoter. Although the HDAC1 protein was downregulated in IFIXalpha-expressing cells, IFIXalpha did not affect HDAC1 mRNA levels. Conversely, a proteasome inhibitor restored the level of HDAC1 protein in IFIXalpha-expressing cells, and the polyubiqutination of HDAC1 was promoted by IFIXalpha, suggesting that HDAC1 is regulated by IFIXalpha through a ubiquitin-proteasome pathway. Together, these data provide novel insights into the tumor-suppressive function of IFIXalpha.

Interferon-inducible protein IFIXalpha1 functions as a negative regulator of HDM2.

The 200-amino-acid repeat (HIN-200) gene family with the hematopoietic interferon (IFN)-inducible nuclear protein encodes highly homologous proteins involved in cell growth, differentiation, autoimmunity, and tumor suppression. IFIX is the newest member of the human HIN-200 family and is often downregulated in breast tumors and breast cancer cell lines. The expression of the longest isoform of IFIX gene products, IFIXalpha1, is associated with growth inhibition, suppression of transformation, and tumorigenesis. However, the mechanism underlying the tumor suppression activity of IFIXalpha1 is not well understood. Here, we show that IFIXalpha1 downregulates HDM2, a principal negative regulator of p53, at the posttranslational level. IFIXalpha1 destabilizes HDM2 protein and promotes its ubiquitination. The E3 ligase activity of HDM2 appears to be required for this IFIXalpha1 effect. Importantly, HDM2 downregulation is required for the IFIXalpha1-mediated increase of p53 protein levels, transcriptional activity, and nuclear localization, suggesting that IFIXalpha1 positively regulates p53 by acting as a negative regulator of HDM2. We found that IFIXalpha1 interacts with HDM2. Interestingly, the signature motif of the HIN-200 gene family, i.e., the 200-amino-acid HIN domain of IFIXalpha1, is sufficient not only for binding HDM2 but also for downregulating it, leading to p53 activation. Finally, we show that IFIX mediates HDM2 downregulation in an IFN-inducible system. Together, these results suggest that IFIXalpha1 functions as a tumor suppressor by repressing HDM2 function.

Antitumor activity of IFIX, a novel interferon-inducible HIN-200 gene, in breast cancer.

We identified IFIX as a new member of the hematopoietic interferon (IFN)-inducible nuclear protein with the 200-amino-acid repeat (HIN-200) family. Six different alternatively spliced forms of mRNA are transcribed from the IFIX gene, which are predicted to encode six different isoforms of IFIX proteins (IFIXalpha1, alpha2, beta1, beta2, gamma1, and gamma2). The IFIX proteins are primarily localized in the nucleus. They share a common N-terminal region that contains a predicted pyrin domain and a putative nuclear localization signal. Unlike IFIXalpha and IFIXbeta, IFIXgamma isoforms do not have the 200-amino-acid signature motif. Interestingly, the expression of IFIX was reduced in most human breast tumors and breast cancer cell lines. Expression of IFIXalpha1, the longest isoform of IFIX, in human breast cancer cell lines reduced their anchorage-dependent and -independent growth in vitro and tumorigenicity in nude mice. Moreover, a liposome-mediated IFIXalpha1 gene transfer suppressed the growth of already-formed tumors in a breast cancer xenograft model. IFIXalpha1 appears to suppress the growth of breast cancer cells in a pRB- and p53-independent manner by increasing the expression of the cyclin-dependent kinase inhibitor p21(CIP1), which leads to the reduction of the kinase activity of both Cdk2 and p34(Cdc2). Together, our results show that IFIXalpha1 possesses a tumor-suppressor activity and suggest IFIXalpha1 may be used as a therapeutic agent in cancer treatment.

A delayed chemically induced tumorigenesis in Brca2 mutant mice.

BRCA2 is a breast cancer susceptibility gene. Germline mutations of BRCA2 account for about 10-30% of familial breast cancer cases. Consistent with its tumor-suppressor activity, BRCA2 plays an important role in DNA repair. To assess the susceptibility of carriers of mutant BRCA2 to tumorigenesis induced by DNA-damaging carcinogens, we generated a Brca2 knockout mouse strain and studied its susceptibility to chemically induced tumorigenesis. Similar to previously reported Brca2 knockout mice, our Brca2-/- embryos die at E8.5-9.5, while the Brca2+/- mice are tumor-free and fertile. Unexpectedly, Brca2+/- mice developed tumors slower than did their wild-type littermates when treated with a potent carcinogen 7,12-dimethylbenz[a]anthracene (DMBA). In vitro experiments showed that Brca2+/- mouse cells and Capan-1 cells, a human pancreatic cancer cell line deficient of BRCA2, were more sensitive to DMBA-induced apoptosis, than were Brca2+/+ mouse cells and a derivative of Capan-1 cells that expressed exogenous wild-type BRCA2, respectively. Our results suggest that enhanced sensitivity of Brca2 mutant cells to DMBA-induced apoptosis at the dose of DMBA we used contributes to the delayed tumorigenesis of Brca2+/- animals. This suggestion may also provide a rational explanation for a previous unexpected finding that cigarette smoking appears to reduce the breast cancer risk of BRCA2 mutation carriers.

Prostate-specific antitumor activity by probasin promoter-directed p202 expression.

p202, an interferon (IFN) inducible protein, arrests cell cycle at G1 phase leading to cell growth retardation. We previously showed that ectopic expression of p202 in human prostate cancer cells renders growth inhibition and suppression of transformation phenotype in vitro. In this report, we showed that prostate cancer cells with stable expression of p202 were less tumorigenic than the parental cells. The antitumor activity of p202 was further demonstrated by an ex vivo treatment of prostate cancer cells with p202 expression vector that showed significant tumor suppression in mouse xenograft model. Importantly, to achieve a prostate-specific antitumor effect by p202, we employed a prostate-specific probasin (ARR2PB) gene promoter to direct p202 expression (ARR2PB-p202) in an androgen receptor (AR)-positive manner. The ARR2PB-p202/liposome complex was systemically administered into mice bearing orthotopic AR-positive prostate tumors. We showed that parenteral administration of an ARR2PB-p202/liposome preparation led to prostate-specific p202 expression and tumor suppression in orthotopic prostate cancer xenograft model. Furthermore, with DNA array technique, we showed that the expression of p202 was accompanied by downregulation of G2/M phase cell-cycle regulators, cyclin B, and p55cdc. Together, our results suggest that p202 suppresses prostate tumor growth, and that a prostate-specific antitumor effect can be achieved by systemic administration of liposome-mediated delivery of ARR2PB-p202.

P202, an interferon-inducible protein, inhibits E2F1-mediated apoptosis in prostate cancer cells.

p202, an interferon (IFN) inducible protein, is a phosphonuclear protein involved in the regulation of cell cycle, apoptosis, and differentiation. E2F1 belongs to the E2F family of proteins that are important cell cycle regulators in promoting cell growth. On the other hand, the deregulated expression of E2F1 also triggers apoptosis independent of p53 status. It has been well documented that p202 is able to inhibit cell growth by binding to E2F1 and abolishing the E2F1-mediated transcriptional activation of S-phase genes. However, it is not known whether E2F1-mediated apoptosis can be counteracted by p202 expression. Here, we show that E2F1-mediated apoptosis induced by the infection of an E2F1-expressing adenoviral vector (Ad-E2F1) was greatly diminished in p202-expressing prostate cancer cells. The E2F1-mediated caspase-3 activation was also reduced in p202-expressing cells infected with Ad-E2F1. Since caspase-3 is one of the E2F1 transcriptional targets, this result is consistent with the ability of p202 to inhibit the transcriptional activity of E2F1. Therefore, our results suggest a possible link between the IFN and E2F pathways in regulating apoptosis.

Proapoptotic and antitumor activities of adenovirus-mediated p202 gene transfer.

PURPOSE AND EXPERIMENTAL DESIGN: p202, a mouse IFN-inducible protein, is a member of the 200-amino acid repeat family. Enforced p202 expression in stable cancer cell lines resulted in growth inhibition in vitro and tumor suppression in vivo. However, to study the immediate effect of p202 and test the potential efficacy of p202 treatment, an efficient gene delivery system for p202 is required. For these purposes, an adenoviral vector expressing the p202 gene (Ad-p202) was generated. We examined the effects of Ad-p202 infection on human breast cancer cells. Furthermore, we tested the efficacy of Ad-p202 treatment on breast and pancreatic cancer xenograft models. RESULTS: We found that Ad-p202 infection induces growth inhibition and sensitizes the otherwise resistant cells to tumor necrosis factor alpha-induced apoptosis. In addition, we demonstrated for the first time that Ad-p202 infection induces apoptosis and that activation of caspases is required for the full apoptotic effect. More importantly, we showed the efficacy of Ad-p202 treatment on breast cancer xenograft models, and this antitumor effect correlated well with enhanced apoptosis in Ad-p202-treated tumors. CONCLUSIONS: We conclude that Ad-p202 is a potent growth-inhibitory, proapoptotic, and tumor-suppressing agent. Ad-p202 may be further developed into an efficient therapeutic agent for human cancer gene therapy.