T.R. Jeffry Evans

Mutant p53 drives metastasis and overcomes growth arrest/senescence in pancreatic cancer

TP53 mutation occurs in 50–75% of human pancreatic ductal adenocarcinomas (PDAC) following an initiating activating mutation in the KRAS gene. These p53 mutations frequently result in expression of a stable protein, p53R175H, rather than complete loss of protein expression. In this study we elucidate the functions of mutant p53 (Trp53R172H), compared to knockout p53 (Trp53fl), in a mouse model of PDAC. First we find that although KrasG12D is one of the major oncogenic drivers of PDAC, most KrasG12D-expressing pancreatic cells are selectively lost from the tissue, and those that remain form premalignant lesions. Loss, or mutation, of Trp53 allows retention of the KrasG12D-expressing cells and drives rapid progression of these premalignant lesions to PDAC. This progression is consistent with failed growth arrest and/or senescence of premalignant lesions, since a mutant of p53, p53R172P, which can still induce p21 and cell cycle arrest, is resistant to PDAC formation. Second, we find that despite similar kinetics of primary tumor formation, mutant p53R172H, as compared with genetic loss of p53, specifically promotes metastasis. Moreover, only mutant p53R172H-expressing tumor cells exhibit invasive activity in an in vitro assay. Importantly, in human PDAC, p53 accumulation significantly correlates with lymph node metastasis. In summary, by using ‘knock-in’ mutations of Trp53 we have identified two critical acquired functions of a stably expressed mutant form of p53 that drive PDAC; first, an escape from KrasG12D-induced senescence/growth arrest and second, the promotion of metastasis.

SEARCH: A Phase III, Randomized, Double-Blind, Placebo-Controlled Trial of Sorafenib Plus Erlotinib in Patients With Advanced Hepatocellular Carcinoma

PURPOSE To compare the clinical outcomes of sorafenib plus either erlotinib or placebo in patients with advanced hepatocellular carcinoma (HCC) in a multicenter, multinational, randomized, phase III trial. PATIENTS AND METHODS Patients with advanced HCC and underlying Child-Pugh class A cirrhosis, who were naive to systemic treatment (N = 720), were randomly assigned to sorafenib plus either erlotinib (n = 362) or placebo (n = 358). The primary end point was overall survival (OS). RESULTS Median OS was similar in the sorafenib plus erlotinib and sorafenib plus placebo groups (9.5 v 8.5 months, respectively; hazard ratio [HR], 0.929; P = .408), as was median time to progression (3.2 v 4.0 months, respectively; HR, 1.135; P = .18). In the sorafenib/erlotinib arm versus the sorafenib/placebo arm, the overall response rate trended higher (6.6% v 3.9%, respectively; P = .102), whereas the disease control rate was significantly lower (43.9% v 52.5%, respectively; P = .021). The median durations of treatment with sorafenib were 86 days in the sorafenib/erlotinib arm and 123 days in the sorafenib/placebo arm. In the sorafenib/erlotinib and sorafenib/placebo arms, the rates of treatment-emergent serious AEs (58.0% v 54.6%, respectively) and drug-related serious AEs (21.0% v 22.8%, respectively) were similar. AEs matched the known safety profiles of both agents, but rates of rash/desquamation, anorexia, and diarrhea were higher in the sorafenib/erlotinib arm, whereas rates of alopecia and hand-foot skin reaction were higher in the sorafenib/placebo arm. Withdrawal rates for AEs during cycles 1 to 3 were higher in the sorafenib/erlotinib arm. CONCLUSION Adding erlotinib to sorafenib did not improve survival in patients with advanced HCC.

Phase III Randomized Trial of Doxorubicin and Docetaxel Versus Doxorubicin and Cyclophosphamide As Primary Medical Therapy in Women With Breast Cancer: An Anglo-Celtic Cooperative Oncology Group Study

PURPOSE To compare the clinical and pathologic response rates of doxorubicin and cyclophosphamide (AC) with doxorubicin and docetaxel (AD) as primary chemotherapy in women with primary or locally advanced breast cancer. PATIENTS AND METHODS Eligible patients with histologically proven breast cancer with primary tumors >/= 3 cm, inflammatory or locally advanced disease, and no evidence of metastases were randomly assigned to receive a maximum of six cycles of either doxorubicin (60 mg/m(2)) plus cyclophosphamide (600 mg/m(2)) administered intravenously (IV) every 3 weeks or doxorubicin (60 mg/m(2)) plus docetaxel (75 mg/m(2)) IV every 3 weeks, followed by surgery on completion of chemotherapy. Results A total of 363 patients were randomly assigned to AC (n = 180) or AD (n = 183). A complete clinical response was observed in 17% and 20% of patients treated with AC and AD, respectively (P = .42). Overall (complete and partial) clinical response rates for AC and AD were 61% and 70%, respectively (P = .06). There was no significant difference in either the pathologic complete response rates in the breast with AC (24%) and AD (21%; P = .61) or in the number of patients with positive axillary nodes at surgery with AC (61%) and AD (66%; P = .28). At a median follow-up of 32 months, there is no significant difference between the two groups for the number of relapses. CONCLUSION In contrast to the positive results reported for sequential docetaxel after AC as primary chemotherapy of breast cancer, our data do not suggest a benefit for simultaneous AD over AC.

MicroRNA Molecular Profiles Associated with Diagnosis, Clinicopathologic Criteria, and Overall Survival in Patients with Resectable Pancreatic Ductal Adenocarcinoma

Purpose: MicroRNAs (miRNA) have potential as diagnostic and prognostic biomarkers and as therapeutic targets in cancer. We sought to establish the relationship between miRNA expression and clinicopathologic parameters, including prognosis, in pancreatic ductal adenocarcinoma (PDAC). Experimental Design: Global miRNA microarray expression profiling of prospectively collected fresh-frozen PDAC tissue was done on an initial test cohort of 48 patients, who had undergone pancreaticoduodenectomy between 2003 and 2008 at a single institution. We evaluated association with tumor stage, lymph node status, and site of recurrence, in addition to overall survival, using Cox regression multivariate analysis. Validation of selected potentially prognostic miRNAs was done in a separate cohort of 24 patients. Results: miRNA profiling identified expression signatures associated with PDAC, lymph node involvement, high tumor grade, and 20 miRNAs were associated with overall survival. In the initial cohort of 48 PDAC patients, high expression of miR-21 (HR = 3.22, 95% CI: 1.21–8.58) and reduced expression of miR-34a (HR = 0.15, 95% CI: 0.06–0.37) and miR-30d (HR = 0.30, 95% CI: 0.12–0.79) were associated with poor overall survival following resection independent of clinical covariates. In a further validation set of 24 patients, miR-21 and miR-34a expression again significantly correlated with overall survival (P = 0.031 and P = 0.001). Conclusion: Expression patterns of miRNAs are significantly altered in PDAC. Aberrant expression of a number of miRNAs was independently associated with reduced survival, including overexpression of miR-21 and underexpression of miR-34a. Summary: miRNA expression profiles for resected PDAC were examined to identify potentially prognostic miRNAs. miRNA microarray analysis identified statistically unique profiles, which could discriminate PDAC from paired nonmalignant pancreatic tissues as well as molecular signatures that differ according to pathologic features. miRNA expression profiles correlated with overall survival of PDAC following resection, indicating that miRNAs provide prognostic utility. Clin Cancer Res; 18(2); 534–45. ©2011 AACR.

Identification of potential biomarkers for measuring inhibition of Src kinase activity in colon cancer cells following treatment with dasatinib

Elevated levels of Src kinase expression have been found in a variety of human epithelial cancers. Most notably in colon cancer, elevated Src expression correlates with malignant potential and is also associated with metastatic disease. Dasatinib (BMS-354825) is a novel, orally active, multi-targeted kinase inhibitor that targets Src family kinases and is currently under clinical evaluation for the treatment of solid tumors. However, the effects of dasatinib on epithelial tumors are not fully understood. We show that concentrations of dasatinib that inhibit Src activity do not inhibit proliferation in 10 of 12 colon cancer cells lines. However, inhibition of integrin-dependent adhesion and migration by dasatinib correlated with inhibition of Src activity, suggesting that dasatinib may have anti-invasive or anti-metastatic activity and antiproliferative activity in epithelial tumors. Using phospho-specific antibodies, we show that inhibition of Src activity in colon cancer cell lines correlates with reduced phosphorylation of focal adhesion kinase and paxillin on specific Src-dependent phosphorylation sites. We have validated the use of phospho-specific antibodies against Src Tyr419 and paxillin Tyr118 as biomarkers of dasatinib activity in vivo. Colon carcinoma–bearing mice treated with dasatinib showed a decrease in both phospho-Src Tyr419 and phospho-paxillin Tyr118 in peripheral blood mononuclear cells, which correlated with inhibition of Src activity in the colon tumors. Thus, peripheral blood mononuclear cells may provide a useful surrogate tissue for biomarker studies with dasatinib using inhibition of Src Tyr419 and paxillin Tyr118 phosphorylation as read-outs of Src activity. [Mol Cancer Ther 2006;5(12):3014–22]

CXCR2 Inhibition Profoundly Suppresses Metastases and Augments Immunotherapy in Pancreatic Ductal Adenocarcinoma.

Summary CXCR2 has been suggested to have both tumor-promoting and tumor-suppressive properties. Here we show that CXCR2 signaling is upregulated in human pancreatic cancer, predominantly in neutrophil/myeloid-derived suppressor cells, but rarely in tumor cells. Genetic ablation or inhibition of CXCR2 abrogated metastasis, but only inhibition slowed tumorigenesis. Depletion of neutrophils/myeloid-derived suppressor cells also suppressed metastasis suggesting a key role for CXCR2 in establishing and maintaining the metastatic niche. Importantly, loss or inhibition of CXCR2 improved T cell entry, and combined inhibition of CXCR2 and PD1 in mice with established disease significantly extended survival. We show that CXCR2 signaling in the myeloid compartment can promote pancreatic tumorigenesis and is required for pancreatic cancer metastasis, making it an excellent therapeutic target.

LKB1 Haploinsufficiency Cooperates With Kras to Promote Pancreatic Cancer Through Suppression of p21-Dependent Growth Arrest

Background & Aims Patients carrying germline mutations of LKB1 have an increased risk of pancreatic cancer; however, it is unclear whether down-regulation of LKB1 is an important event in sporadic pancreatic cancer. In this study, we aimed to investigate the impact of LKB1 down-regulation for pancreatic cancer in mouse and human and to elucidate the mechanism by which Lkb1 deregulation contributes to this disease. Methods We first investigated the consequences of Lkb1 deficiency in a genetically modified mouse model of pancreatic cancer, both in terms of disease progression and at the molecular level. To test the relevance of our findings to human pancreatic cancer, we investigated levels of LKB1 and its potential targets in human pancreatic cancer. Results We definitively show that Lkb1 haploinsufficiency can cooperate with oncogenic KrasG12D to cause pancreatic ductal adenocarcinoma (PDAC) in the mouse. Mechanistically, this was associated with decreased p53/p21-dependent growth arrest. Haploinsufficiency for p21 (Cdkn1a) also synergizes with KrasG12D to drive PDAC in the mouse. We also found that levels of LKB1 expression were decreased in around 20% of human PDAC and significantly correlated with low levels of p21 and a poor prognosis. Remarkably, all tumors that had low levels of LKB1 had low levels of p21, and these tumors did not express mutant p53. Conclusions We have identified a novel LKB1-p21 axis that suppresses PDAC following Kras mutation in vivo. Down-regulation of LKB1 may therefore serve as an alternative to p53 mutation to drive pancreatic cancer in vivo.

Phase I Dose-Escalation and Pharmacokinetic Study of Dasatinib in Patients with Advanced Solid Tumors

Purpose: To determine the maximum tolerated dose, dose-limiting toxicity (DLT), and recommended phase II dose of dasatinib in metastatic solid tumors refractory to standard therapies or for which no effective standard therapy exists. Experimental Design: In this phase I, open-label, dose-escalation study, patients received 35 to 160 mg of dasatinib twice daily in 28-day cycles either every 12 hours for 5 consecutive days followed by 2 nontreatment days every week (5D2) or as continuous, twice-daily (CDD) dosing. Results: Sixty-seven patients were treated (5D2, n = 33; CDD, n = 34). The maximum tolerated doses were 120 mg twice daily 5D2 and 70 mg twice daily CDD. DLTs with 160 mg 5D2 were recurrent grade 2 rash, grade 3 lethargy, and one patient with both grade 3 prolonged bleeding time and grade 3 hypocalcemia; DLTs with 120 mg twice daily CDD were grade 3 nausea, grade 3 fatigue, and one patient with both grade 3 rash and grade 2 proteinuria. The most frequent treatment-related toxicities across all doses were nausea, fatigue, lethargy, anorexia, proteinuria, and diarrhea, with infrequent hematologic toxicities. Pharmacokinetic data indicated rapid absorption, dose proportionality, and lack of drug accumulation. Although no objective tumor responses were seen, durable stable disease was observed in 16% of patients. Conclusion: Dasatinib was well tolerated in this population, with a safety profile similar to that observed previously in leukemia patients, although with much less hematologic toxicity. Limited, although encouraging, preliminary evidence of clinical activity was observed. Doses of 120 mg twice daily (5D2) or 70 mg twice daily (CDD) are recommended for further studies in patients with solid tumors. (Clin Cancer Res 2009;15(19):6232–40)

Dasatinib Inhibits the Development of Metastases in a Mouse Model of Pancreatic Ductal Adenocarcinoma

BACKGROUND & AIMS Pancreatic ductal adenocarcinoma (PDAC) is a highly invasive and metastatic disease for which conventional treatments are of limited efficacy. A number of agents in development are potential anti-invasive and antimetastatic agents, including the Src kinase inhibitor dasatinib. The aim of this study was to assess the importance of Src in human PDAC and to use a genetically engineered mouse model of PDAC to determine the effects of dasatinib on PDAC progression. METHODS Src expression and activity was measured by immunohistochemistry in 114 human PDACs. Targeting expression of Trp53(R172H) and Kras(G12D) to the mouse pancreas results in the formation of invasive and metastatic PDAC. These mice were treated with dasatinib, and disease progression monitored. Cell lines were derived from mouse PDACs, and in vitro effects of dasatinib assessed. RESULTS Src expression and activity were up-regulated in human PDAC and this correlated with reduced survival. Dasatinib inhibited the migration and invasion of PDAC cell lines, although no effects on proliferation were seen at concentrations that inhibited Src kinase activity. In addition, dasatinib significantly inhibited the development of metastases in Pdx1-Cre, Z/EGFP, LSL-Kras(G12D/+), LSL-Trp53(R172H/+) mice. However, there was no survival advantage in the dasatinib-treated animals owing to continued growth of the primary tumor. CONCLUSIONS This study confirms the importance of Src in human PDAC and shows the usefulness of a genetically engineered mouse model of PDAC for assessing the activity of potential antimetastatic agents and suggests that dasatinib should be evaluated further as monotherapy after resection of localized invasive PDAC.

Intravital FLIM-FRET Imaging Reveals Dasatinib-Induced Spatial Control of Src in Pancreatic Cancer

Cancer invasion and metastasis occur in a complex three-dimensional (3D) environment, with reciprocal feedback from the surrounding host tissue and vasculature-governing behavior. In this study, we used a novel intravital method that revealed spatiotemporal regulation of Src activity in response to the anti-invasive Src inhibitor dasatinib. A fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET) Src biosensor was used to monitor drug-targeting efficacy in a transgenic p53-mutant mouse model of pancreatic cancer. In contrast to conventional techniques, FLIM-FRET analysis allowed for accurate, time-dependent, live monitoring of drug efficacy and clearance in live tumors. In 3D organotypic cultures, we showed that a spatially distinct gradient of Src activity exists within invading tumor cells, governed by the depth of penetration into complex matrices. In parallel, this gradient was also found to exist within live tumors, where Src activity is enhanced at the invasive border relative to the tumor cortex. Upon treatment with dasatinib, we observed a switch in activity at the invasive borders, correlating with impaired metastatic capacity in vivo. Src regulation was governed by the proximity of cells to the host vasculature, as cells distal to the vasculature were regulated differentially in response to drug treatment compared with cells proximal to the vasculature. Overall, our results in live tumors revealed that a threshold of drug penetrance exists in vivo and that this can be used to map areas of poor drug-targeting efficiency within specific tumor microenvironments. We propose that using FLIM-FRET in this capacity could provide a useful preclinical tool in animal models before clinical translation.