Lingle, Wilma L.

Abstract Background: We recently reported a 49% RECIST response rate in conjunction with a phase 2 clinical trial of the multi-targeted kinase inhibitor pazopanib in differentiated thyroid cancers (DTC; Lancet Oncology 2010, 11:962-72), prompting a search for synergistic pazopanib-containing doublets in thyroid cancer cell lines. Methods and Results: Among all explored pazopanib-containing combinations, paclitaxel and pazopanib produced the greatest combined cytotoxic effects (continuous exposure pazopanib, 24 h paclitaxel; Combination Index, CI, at IC90=0.3). Interestingly, analogous synergy resulted when combining pazopanib with other antimicrotubule agents including other taxanes (docetaxel), Vinca alkyloids (vincristine) and epothilones (ixabepilone) – suggesting a class effect. Further, we found this combination to be synergistic in multiple cancer cell lines including DTC, medullary and anaplastic thyroid cancer lines, and in the A549 lung cancer line. In pursuit of underlying mechanism(s), we noted that the combination produced a greater fraction of cells in G2/M phase of the cell cycle and increased apoptosis in comparison to paclitaxel alone, prompting us to examine whether pazopanib might potentiate the effects of paclitaxel on mitosis. Analysis of cell fate resulting from pazopanib and paclitaxel alone and in combination using time-lapsed video microscopy demonstrated that as low as 2.5 μM pazopanib augmented paclitaxel-induce mitotic catastrophe by 2.4 fold, while inducing no appreciable cell death alone. As pazopanib is somewhat promiscuous in inhibition of kinases, we hypothesized that observed combined effects with antimicrotubule agents might be attributed to its inhibition of cell cycle by targeting CDKs or mitosis by targeting aurora kinases. Inhibition of CDKs 1 and 2 required pazopanib concentrations 1000-times those required to inhibit its primary kinase targets, VEGF-Rs; whereas aurora kinases A and B were inhibited at much lower concentrations (IC50s, 500-1000 nM). Hence, we pursued shRNA aurora A knockdown experiments: potentiation of pazopanib effects were observed in knockdown clones, suggesting that pazopanib single-agent effects are at least in part mediated via aurora A inhibition. Moreover, augmentation of paclitaxel effects were achieved in knockdown clones that rivaled those observed for the pazopanib/paclitaxel combination. Additionally, pazopanib/paclitaxel synergy was reduced in aurora A knockdown clones (CI at IC80: vector<0.5, knockdown clones >1.0), indicating that aurora A inhibition by pazopanib is contributory to paclitaxel/pazopanib synergy. Conclusions: Pazopanib combines with antimicrotubule agents to produce cytotoxic synergy associated with augmentation of mitotic catastrophe that appears at least in part mediated by its ability to inhibit aurora kinases. Supported in part by CA125750. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3597. doi:10.1158/1538-7445.AM2011-3597

Abstract Breast cancer is the most common form of cancer diagnosed and the second leading cause of cancer death among women in the United States. Cancer metastases are the major cause of morbidity and mortality among breast cancer patients. The central importance of the HER2/RAS signaling pathway has been well established in promoting tumor growth, invasion and metastasis of human breast cancer. Even though oncogenic K-RAS mutations are rare in breast cancer, activation of the growth-promoting ERBB/RAS pathway has been consistently documented in high-grade breast tumors. As such, novel approaches to inhibit activated HER2/RAS signals constitute important measures to block tumor growth and metastasis in mammary tumors. In this study, instead of targeting an upstream signaling component such as HER2/EGFR/RAS, we targeted the most downstream signaling module identified in the ERBB/RAS pathway – the SIAH proteolytic machinery. SIAH is an evolutionarily conserved RING E3 ligase that is a critical “gatekeeper” required for RAS signal transduction in human cancer cells. SIAH is specifically expressed in tumor cells in 120 human breast cancer patients examined so far. The increased SIAH expression is correlated with increased grades and aggressiveness of human breast cancer. We asked whether inhibiting the gatekeeper function of SIAH would be effective in blocking mammary tumorigenesis and metastasis in human breast cancer. Our results indicated that SIAH2-deficiency successfully abolished tumor growth of three breast cancer cell lines (MDA-MB-231, MDA-MB-435 and MDA-MB-468) in soft agar assays as well as in athymic nude mice. Importantly, by inhibiting SIAH2 function in highly aggressive human breast cancer cells, we are able to completely block tumorigenesis and metastasis of two highly invasive and metastatic human breast cancer cell lines (MDA-MB-231 and MDA-MB-435). These results suggest that SIAH2 may be an attractive and new drug target for novel breast cancer therapy. Developing anti-SIAH molecules will aid in expanding our arsenal of effective anticancer therapies. By attacking the growth-promoting HER2/EGFR/RAS pathway at multiple signaling modules simultaneously – a multi-pronged synergistic inhibition at upstream (HER2/EGFR membrane receptors), midstream (RAS/RAF/MEK/mTOR) and downstream (SIAH E3 ligase) signaling modules in the HER2/EGFR/RAS pathway in parallel, we may be in a position to halt the genesis, progression and metastasis of human breast cancer. Thus, SIAH-dependent proteolysis may be an Achilles heel for breast cancer. SIAH inhibition may represent an innovative and logical way to inhibit HER2/EGFR/K-RAS activation, alleviate tumor burdens, halt cancer metastasis, and improve the efficacy for the future treatment of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1659. doi:10.1158/1538-7445.AM2011-1659

Abstract Src transduces activated HER tyrosine kinase and phosphorylates HER to further increase its activity. Src also is involved in osteoclast differentiation. We hypothesized that combining a HER tyrosine kinase inhibitor with a Src inhibitor would result in increased therapeutic activity. To address this hypothesis we performed a phase I trial of D and L to: 1)determine the maximally tolerated dose (MTD) of D combined with L; 2)describe the toxicities associated with this combination; 3)assess the pharmacokinetic (PK) interaction of L and D; 4) assess the effect of the L and D combination on circulating tumor cells (CTC) and on osteoclast precursor activation; 5)study the association of toxicity and/or tumor response with PK, and/or pharmacodynamic parameters; and 6)describe the responses of this combination. Eligibility criteria included ≥ 18 years of age, incurable solid tumors not amenable to other therapies, ANC ≥ 1500/ L, PLT ≥ 100,000/ L, total bilirubin ≤ ULN, AST ≤ 3 × ULN or AST ≤5 × ULN if liver involvement, creatinine ≤ 1.5 × ULN, Hgb ≥ 9.0 g/dL, K+ and Mg++ WNL, life expectancy ≥ 12 weeks, ejection fraction > 50%, ECOG PS 0–2, and able to swallow pills whole. In addition to standard phase I exclusion criteria patients could not have received erlotinib, sunitinib, sorafenib, gefitinib, imatinib ≤ 4 weeks prior to registration. Each agent was administered once daily on a 28 day schedule. At the MTD patients were treated with D day 1, and day 9–28 and L day 2–28 to assess PK interaction. Dose levels (mg) were #1 D 70, L 1000, #2 D 100, L 1000, #3 D 140, L 1000. 12 patients were entered on the dose escalation portion of the trial and 15 were entered at the MTD. 5 patients entered at the MTD are too early to be fully evaluable. Characteristics of evaluable patients were: median age, 63; 46% female; PS 0 (64%) 1 (27%), 1 (9%); ≥ 3 prior chemotherapies (59%). DLT was seen in 2 patients at # 3 (1 patient, grade 3 hypermagnesemia, 1, grade 3 rash) and 1 patient at # 2 (grade 3 dyspnea). The MTD was determined to be # 2. No grade 4 or 5 events have been observed in this study. 6 patients had stable disease in the dose escalation cohort. 1 (RCC) was stable disease for 6 cycles, 2 (colorectal) were stable for 5 cycles, 1 (esophagus) was stable for 3 cycles, and 2 (GIST & leiomyosarcoma) were stable for 2 cycles. Analysis of drug levels, CTCs and circulating osteoclast precursors is ongoing. There was a consistent increase in the percentage of CD11b (monocyte/macrophage/osteoclast lineage marker) positive cells in circulation as treatment progressed. However, the late osteoclast lineage marker c-Fms was not similarly increased, suggesting that the elevated CD11b reflects an inflammatory response and not activation of osteoclastogenesis. D and L is a tolerable combination at nearly full doses of each drug. Funded by a grant from Glaxo Smith Kline and Bristol Myers Squibb. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B56.

Abstract Purpose: To investigate the associations between baseline and posttreatment circulating tumor cell (CTC) gene expression and outcome of patients enrolled in four North Central Cancer Treatment Group metastatic breast cancer (MBC) trials in which specimens were shipped (at 4°C) from community-based sites to a reference laboratory (Mayo Clinic, Rochester, MN). Experimental Design: Blood was collected at treating sites from MBC patients before (baseline), during, and at the end of treatment with erlotinib + gemcitabine (N0234), sorafenib (N0336), irinotecan + cetuximab (N0436), or paclitaxel-poliglumex + capecitabine (N0437). CTCs from 10 mL of EDTA blood were enriched with CD45 depletion, 24 to 30 hours postblood collection. Reverse transcription/quantitative PCR was used to determine cytokeratin-19 (CK19) and mammaglobin (MGB1) mRNA levels in CTCs from up to 13 (N0234), 16 (N0336), 18 (N0436), and 39 (N0437) patients. The gene expressions were normalized to β2-microglobulin and calibrated to healthy blood using the 2–ΔΔCq algorithm; positivity was defined as 2 or more. Results: CK19+mRNA cells were detected in 56% to 75% and MGB1+mRNA cells in 23% to 38% of 86 patients at baseline. CK19+mRNA cells were detected in 30% to 67% and MGB1+mRNA cells in 14% to 64% of 110 postbaseline serial samples. The presence of baseline CK19+mRNA cells (P = 0.01) but not MGB1+mRNA cells (P = 0.14) was significantly associated with shorter overall survival. A decrease in MGB1+mRNA levels (baseline-week 8) seemed to be associated with clinical response (P = 0.05). Conclusions: CTC gene expression analysis conducted by a reference laboratory is feasible when blood is collected from treating sites and processed 24 to 30 hours postcollection. The presence of baseline CK19+mRNA CTCs was associated with poor prognosis; a decrease in MGB1+mRNA CTCs may help predict response to therapy of MBC patients. Clin Cancer Res; 17(22); 7183–93. ©2011 AACR.

We used a novel adaptation of methods for microtubule polymerization in vitro to assess the MTOC activity of centrosomes in frozen-sectioned tissues. Remarkably, centrosomes of tissue sections retain the ability to nucleate microtubules even after several years of storage as frozen tissue blocks. Adaptations of these methods allow accurate counts of microtubules from individual cells and the quantitative estimation the MTOC activity of the intact tissue. These methods can be utilized to characterize MTOC activity in normal and diseased tissues and in particular tissues at different stages of development.

Abstract Lung cancers are often characterized by numerical chromosome instability. Hexavalent chromium (Cr(VI)) is a well-known human lung carcinogen; however, its carcinogenic mechanism remains unknown. Studies show that solubility plays a key role in its carcinogenicity, with the particulate, or insoluble, compounds being the most potent carcinogens. Epidemiological and whole animal studies pinpoint particulate zinc chromate as the most potent Cr(VI) carcinogen; however, few studies have investigated its carcinogenic mechanism. Therefore, the goal of this study was to determine if zinc chromate induces numerical chromosome instability in human lung cells. We found that exposure to zinc chromate for 24 h did not induce aneuploidy but longer exposures induced both concentration- and time-dependent increases in aneuploid metaphases. For example, exposure to 0.1, 0.15 and 0.2 ug/cm2 zinc chromate for 120 h induced 28, 40 and 44 percent aneuploid metaphases, respectively, with increases in hypodiploid, hyperdiploid and tetraploid metaphases. Consistent with the aneuploidy data, a 24 h exposure to zinc chromate did not induce centrosome amplification but more chronic exposures induced concentration- and time-dependent increases in centrosome amplification in both interphase and mitotic cells. Exposure to 0.1, 0.15 and 0.2 ug/cm2 zinc chromate for 120 h induced centrosome amplification in 13.9, 18 and 21.3 percent of interphase cells and 21, 33 and 46 percent of mitotic cells. In order to determine the mechanism of centrosome amplification, we investigated the number of centrioles in each centrosome and found that cells with amplified centrosomes exhibited centrosomes with normal centriole pairs and centrosomes with centriolar defects indicating multiple mechanisms are involved in zinc chromate-induced centrosome amplification. Lastly, we found that chronic exposure to zinc chromate induced a prolonged G2 arrest. All together, these data indicate that chronic exposure to zinc chromate induces numerical chromosome instability via centrosome amplification in human lung cells. This work was supported by NIEHS grant ES016893 (J.P.W.), cooperative agreement #EP-08-01 through the Maine Space Grant Consortium (J.P.W.), EPA GRO Fellowship MA-91685401 (A.L.H.) and the Maine Center for Toxicology and Environmental Health at the University of Southern Maine. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3473.

Abstract Breast cancer is the most common form of cancer diagnosed and the second leading cause of cancer death among women in the United States. Cancer metastases are the major cause of morbidity and mortality among breast cancer patients. The central importance of the HER2/Neu/EGFR/RAS signaling pathway has been well established in the growth, invasion and metastasis of human breast cancer, even though oncogenic RAS mutations are rare in breast cancer, activation of the tumor-promoting ERBB/RAS pathway has been consistently documented in high-grade breast tumors. As such, novel approaches to inhibit activated ERBB/RAS signals constitute important measures to block tumor growth and metastasis in mammary tumors. In this study, instead of targeting an upstream signaling component such as HER2/EGFR/RAS, we targeted the most downstream signaling module identified in the RAS pathway - the SIAH proteolytic machinery. SIAH is an evolutionarily conserved RING E3 ligase that is a critical “gatekeeper” required for RAS signal transduction in human cancer cells. SIAH is specifically expressed in tumor cells in 120 human breast cancer patients examined so far. The increased SIAH expression is correlated with increased grades and aggressiveness of human breast cancer. We then asked whether inhibiting the gatekeeper function of SIAH would be effective in blocking mammary tumorigenesis and metastasis in human breast cancer. Our results indicated that SIAH2-deficiency successfully abolished tumor growth of three breast cancer cell lines (MDA-MB-231, MDA-MB-435 and MDA-MB-468) in soft agar assays as well as in athymic nude mice. Importantly, by inhibiting SIAH2 function in highly aggressive human breast cancer cells, we are able to completely block tumorigenesis and metastasis of two highly invasive and metastatic human breast cancer cell lines (MDA-MB-231 and MDA-MB-435). These findings suggest that SIAH2 may be an attractive new therapeutic target for novel breast cancer therapy and developing anti-SIAH molecules will aid in expanding our arsenal of effective anticancer therapies. More effective breast cancer treatments may be obtained by multi-pronged synergistic inhibitions at upstream (HER2/EGFR membrane receptors), midstream (RAS/RAF/MEK/mTOR) and downstream (SIAH E3 ligase) signaling modules in the HER2/EGFR/RAS pathway in parallel. Thus, by attacking the growth-promoting ERBB/RAS pathway at multiple signaling modules simultaneously, we may be in a great position to halt the genesis, progression and metastasis of human breast cancer. If successful, this study has the promise to be translated to the cancer clinics to alleviate tumor burdens, inhibit cancer metastasis, improve the efficacy for the treatment of breast cancer, and in particularly help cancer patients with metastatic diseases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4518.

Abstract HSulf-1 modulates the sulfation states of heparan sulfate proteoglycans critical for heparin binding growth factor signaling. In the present study, we show that HSulf-1 is transcriptionally deregulated under hypoxia in breast cancer cell lines. Knockdown of HIF-1α rescued HSulf-1 downregulation imposed by hypoxia, both at the RNA and protein levels. Chromatin immunoprecipitation with HIF-1α and HIF-2α antibodies confirmed recruitment of HIF-α proteins to the two functional hypoxia-responsive elements on the native HSulf-1 promoter. HSulf-1 depletion in breast cancer cells resulted in an increased and sustained bFGF2 (basic fibroblast growth factor) signaling and promoted cell migration and invasion under hypoxic conditions. In addition, FGFR2 (fibroblast growth factor receptor 2) depletion in HSulf-1–silenced breast cancer cells attenuated hypoxia-mediated cell invasion. Immunohistochemical analysis of 53 invasive ductal carcinomas and their autologous metastatic lesions revealed an inverse correlation for the expression of HSulf-1 to CAIX in both the primary tumors (P ≥ 0.0198) and metastatic lesions (P ≥ 0.0067), respectively, by χ2 test. Finally, HSulf-1 expression levels in breast tumors by RNA in situ hybridization showed that high HSulf-1 expression is associated with increased disease-free and overall survival (P ≥ 0.03 and P ≥ 0.0001, respectively). Collectively, these results reveal an important link between loss of HSulf-1 under hypoxic microenvironment and increased growth factor signaling, cell migration, and invasion. Cancer Res; 71(6); 2152–61. ©2011 AACR.

Purpose Findings from the human epidermal growth factor receptor 2 (HER2) –positive National Surgical Adjuvant Breast and Bowel Project (NSABP) B31 trial suggested that MYC/HER2 coamplification (> 5.0 copies/nucleus) was associated with additional benefit from adjuvant trastuzumab in patients with early-stage breast cancer. To further explore this relationship, we investigated associations between MYC amplification and disease-free survival (DFS) in a similar adjuvant trastuzumab HER2-positive breast cancer trial—North Central Cancer Treatment Group (NCCTG) N9831. Patients and Methods This analysis included 799 patients randomly assigned to receive chemotherapy alone or with concurrent trastuzumab on N9831. Fluorescence in situ hybridization (FISH) was performed by using a dual-probe mixture for MYC and centromere 8 (MYC:CEP8) on tissue microarrays. MYC amplification was prespecified as MYC:CEP8 ratio > 2.2 or average MYC copies/nucleus > 5.0. Exploratory variables included polysomy 8. Results In comparing DFS (median follow-up, 4.0 years) between treatments, patients with MYC:CEP8 ratio ≤ 2.2 (n = 618; 77%) and > 2.2 (n = 181; 23%) had hazard ratios (HRs) of 0.46 (P < .001) and 0.67 (P = .33), respectively (interaction P = .38). Patients with MYC copies/nucleus ≤ 5.0 (n = 534; 67%) and > 5.0 (n = 265; 33%) had HRs of 0.52 (P = .002) and 0.48 (P = .02), respectively (interaction P = .94). Patients with MYC:CEP8 ratio < 1.3 with normal chromosome 8 copy number (n = 141; 18%) and ≥ 1.3 or < 1.3 with polysomy 8 (n = 658; 82%) had HRs of 0.66 (P = .28) and 0.44 (P < .001), respectively (interaction P = .23). Patients with MYC copies/nucleus < 2.5 (n = 130; 16%) and ≥ 2.5 (n = 669; 84%) had HRs of 1.07 (P = .87) and 0.42 (P < .001), respectively (interaction P = .05). Conclusion We did not confirm the B31 association between MYC amplification and additional trastuzumab benefit. Exploratory analyses revealed potential associations between alternative MYC/chromosome 8 copy number alterations and differential benefit of adjuvant trastuzumab.

Abstract Background: Gene expression profiling is an important tool in prediction, prognosis, and cancer modeling. The DASL® assay (cDNA –mediated Annealing, extension, Selection, and Ligation; Illumina; San Diego, CA) is a gene expression profiling system designed for degraded RNAs such as those derived from formalin-fixed paraffin-embedded (FFPE) tumor samples. FFPE tumor samples are invaluable resources for cancer research, being the most widely available materials for which patient outcomes are known. Purpose: The objective of this study was to evaluate the performance of Illumina's Whole Genome DASL platform (24,526; 24K) at distinguishing HER2-positive (HER2+) from HER2-negative (HER2-) FFPE breast tumors. Methods: We measured the relative gene expression of biological and technical replicates using nine HER2+ and 11 HER2- primary breast tumors from patients that were all node-positive and estrogen receptor-positive. The HER2 status of the tumors was determined using the Dako Hercept Test following the 2007 ASCO/CAP guidelines for HER2 positivity. The non-background corrected probe-expression data were exported from Illumina BeadStudio software and normalized using the software R and the function fastlo. Log2 transformed expression values were used to obtain the relative gene expression per probe. Spearman correlations were calculated between both types of replicates. Unequal-variance t-tests were conducted to test for differences in expression levels between HER2+ and HER2- tumors. An overall ERBB2 gene expression level was calculated by taking the average of the three ERBB2 probes for each tumor across all replicates. To simulate the possible outcome if only one replicate of each tumor was measured for gene expression, differential expression was tested for all possible combinations of single replicate probe expression values. Results: Spearman correlations for technical and biological replicates within tumor samples ranged from 0.9804 – 0.9946, and 0.9822 – 0.9945, respectively. The overall ERBB2 gene expression was significantly lower in HER2- tumors than in HER2+ tumors (p = 0.0000018). The top five most differentially expressed genes (p &lt; 0.0005) included ERBB2, GRB7, C17ORF37, PERLD1, and ASF1B. For each of the three ERBB2 probes, there were 65,536 possible ways to combine the individual probe expression values. The p-values for testing differential expression in each of these 65,536 combinations of HER2+ tumors versus HER2- tumors ranged from 0.00029 – 0.0091 for ILMN_1717902, 0.0000078 – 0.00026 for ILMN_1728761, and 0.000000002 – 0.0000032 for ILMN-2352131. Conclusions: Illumina's Whole Genome Platform provided highly reproducible gene expression values among biological and technical replicates of breast tumors. This platform also demonstrated significant differential ERBB2 gene expression between HER2+ and HER- breast tumors, which correctly classified the breast tumors as HER2+ and HER2-. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6127.