Citations

Abstract A better understanding of the antagonistic interaction and cross-talk between the pivotal oncogenic and tumor suppressor signaling pathways, and the concerted cancer signaling networks that control tumorigenesis and metastasis should give rise to novel therapeutic interventions. Hyperactivated ERBB/RAS is known to drive neoplastic transformation, tumorigenesis and metastasis in a large percentage of human cancers, and thus finding novel measures to countervail activated EGFR/HER2/RAS signals to reverse tumorigenesis and metastasis remains an important goal in cancer biology. Instead of targeting an upstream signaling component, we targeted the most downstream signaling module in the ERBB/RAS pathway, the SIAH-dependent proteolytic machinery. SIAHs are evolutionarily conserved RING E3 ligases that are the critical downstream “gatekeeper” signaling modules in the RAS signal transduction pathway. We show that human SIAH E3 ligase is upregulated in proliferating tumor cells in human tumor specimens, and that proper SIAH function is required for RAS signal transduction. By inhibiting SIAH2 E3 ligase, we have blocked both tumorigenesis and metastasis in eleven of the most aggressive human cancer cell lines known in athymic nude mice. Our observations highlight the important roles that SIAH2 plays in RAS-dependent tumorigenesis and metastasis in human cancer. In this study, we aim to identify the molecular mechanism underlying SIAH2 deficiency-mediated growth inhibition in human cancer cells. We have found that SMAD2/3/4 - the essential downstream mediators of TGFβ signaling, are novel SIAH2 substrates in that SIAH2 physically interacts with, ubiquitinates and degrades SMAD2/3/4 (not SMAD1). The identification of SIAH2-mediated SMAD2/3/4 modulation may provide an additional molecular mechanism to explain the well documented, but less understood RAS-TGFβ antagonism described in normal development, tumorigenesis and metastasis. We propose that the tumor promoting activity of SIAH2 may be mediated through the degradation of SMAD2/3/4 proteins which enhancing the growth-promoting activity of the RAS pathway while suppressing the growth inhibitory action of the TGFβ pathway. Since the substrate specificity of ubiquitin-mediated proteolysis is primarily determined by the E3 ligase, our results provide new insights into the role of altered proteolysis in mediating the RAS-TGFβ antagonism and cross-talk observed in the genesis and progression of human cancers. 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 5073.

Abstract The dismal prognosis of patients diagnosed with invasive and metastatic cancer points to our limited arsenal of effective anticancer therapies. The central importance of EGFR/HER2/RAS pathway activation has been well established in neoplastic transformation and tumorigenesis. An important goal in cancer biology is to identify novel and effective means to inhibit activated EGFR/HER2/K-RAS signals and reverse malignant transformation, tumor growth and cancer metastasis. SIAHs are the human homologs of Drosophila Seven-In-Absentia (SINA), an evolutionarily conserved RING E3 ligase that is an essential downstream signaling module and critical “gatekeeper” required for RAS signal transduction in human cancer cells. By targeting the most downstream signaling module identified in the RAS pathway - the SIAH2 proteolytic machinery, we are able to block tumorigenesis and metastasis in multiple of the most aggressive forms of human cancer cells known in athymic nude mice. Although we are fully cognizant of the systemic limitation in the xenograft models and incomplete mimicry of the full-spectrum metastatic processes observed in athymic nude mice, we are encouraged by the impressive anti-SIAH2-based tumor suppression in these preclinical studies. In this study, we aim to identify the molecular mechanism(s) of SIAH2 action in human cancer cells. To delineate SIAH2 function in cancer, through a proteomic approach, we identified three endogenous LIM domain proteins, TRIP6/FHL2/LPXN, as the bona fide SIAH2-interacting proteins isolated from the most aggressive human cancer cell lines (A549, MiaPaCa and MDA-MB-435 cells) in parallel. We provide extensive biochemical evidence to show that TRIP6/FHL2/LPXN are bona fide SIAH2 substrates. These new SIAH2 substrates can successfully rescue cell motility and viability defects observed in SIAH2-deficient cancer cells, suggesting that these focal adhesion proteins may represent one of many diverse signaling modules that function downstream of SIAH2 in mediating aspects of RAS-dependent signaling in normal development and cancer. Our results suggest In response to ERBB/RAS activation, SIAH2 E3 ligase modulates cell junction, adhesion and migration by targeting focal adhesion proteins for ubiquitin-mediated proteolysis to promote cell migration, tumorigenesis and metastasis in human cancer cells. The identification of SIAH2-mediated focal adhesion and cell junction modulation may have important implications in cancer biology to explain the well-documented modulations in focal adhesion, cell junction and motility in response to ERBB/RAS activation in human cancer cells. 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 4808.

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.

Background Telephone quitlines that provide counseling support are efficacious in helping cigarette smokers quit and have been widely disseminated; currently, they are underused. Surgery represents a teachable moment for smoking cessation, which can benefit surgical outcomes; however, few surgical patients receive smoking cessation interventions. This study developed and tested a clinician-delivered intervention to facilitate quitline use by adult patients scheduled for elective surgery. Methods After formative work involving patients and clinicians, a brief intervention was designed to facilitate telephone quitline use. It was then evaluated in a randomized trial of 300 adults scheduled for elective surgery. A control standard brief stop-smoking intervention served as a comparator, with both interventions delivered by clinicians. The primary outcome was the use rate of a quitline accessed through a dedicated toll-free telephone number, with use defined as completing at least one full counseling session. Secondary outcomes included self-reported abstinence from cigarettes at 30 and 90 days postoperatively. Results Subject characteristics were similar between the two groups. Records from the designated quitline documented that 29 of 149 subjects (19.5%) in the quitline intervention group and 0 of 151 subjects in the control group completed the first full counseling session (P < 0.0001). There were no significant differences in the self-reported point-prevalent and continuous abstinence rates between groups at either 30 or 90 days postoperatively, although rates tended to be higher in the quitline intervention group. Conclusions Clinicians can effectively facilitate quitline use by surgical patients. Further work is necessary to evaluate the efficacy of this approach in terms of long-term abstinence from cigarette smoking.

Abstract Sulindac, atorvastatin, or prebiotic dietary fiber may reduce colorectal cancer (CRC) risk. However, clinical trial data are currently limited. We conducted a randomized, phase II chemoprevention trial involving subjects 40 years or older, with previously resected colon cancer or multiple/advanced colorectal adenomas. Magnification chromoendoscopy (MCE) was performed to identify and characterize rectal aberrant crypt foci (ACF); eligibility criteria required five or more rectal ACFs at baseline. Intervention assignments were as follows: (a) atorvastatin 20 mg qd; (b) sulindac 150 mg bid; (c) oligofructose-enriched inulin (as ORAFTI®Synergy1) 6 gm bid; or (d) control (maltodextrin) 6 gm bid, for 6 months. Percent change in rectal ACF number (%ΔACF) within arm was the primary endpoint. Secondary endpoints included changes in proliferation (Ki67) and apoptosis (caspase-3), as measured from normal mucosa biopsy samples. Among 85 eligible randomized subjects, 76 (86%) completed the trial per protocol. The median (range) of rectal ACF was 9 (5–34) and 8 (0–37) at baseline and postintervention, respectively. The median (SD) for %ΔACF was 5.6 (−69% to 143%), −18.6 (−83% to 160%), −3.6 (−88% to 83%), and −10.0 (−100% to 117%) in the atorvastatin, sulindac, ORAFTI®Synergy1 and control arms, respectively. Neither within-arm (P = 0.12–0.59) nor between-arm (P = 0.30–0.92) comparisons of %ΔACF were statistically significant. The active and control interventions also seemed to have similar effects on mucosal proliferation and apoptosis (P &gt; 0.05 for each comparison). Data from this multicenter, phase II trial do not provide convincing evidence of CRC risk reduction from 6-month interventions with atorvastatin, sulindac, or ORAFTI®Synergy1, although statistical power was limited by the relatively small sample size. Cancer Prev Res; 4(2); 259–69. ©2011 AACR.

Abstract BackgroundRIBBON-1, a Phase III, multicenter, randomized, placebo-controlled trial in patients with previously untreated MBC was designed to evaluate the efficacy and safety of adding bevacizumab (B) to chemotherapy regimens including capecitabine (Cape; n=615); a taxane (T; n=307), or an anthracycline (Anth; n=315) compared with chemotherapy alone. Pre-specified analyses of progression-free survival PFS) in the Cape and pooled T/Anth cohorts, the primary endpoints of the study were increased upon addition of B. In the Cape cohort, median PFS increased from 5.7 to 8.6 mo (HR=0.69, p-value=0.0002) and in the T/Anth cohort, it increased from 8.0 to 9.2 mo (HR=0.64; p-value&lt;0.0001).MethodsRECIST was used to determine objective response rate (ORR). Patients without a post-baseline tumor assessment were considered non-responders. The primary analysis for the ORR and the duration of response was performed using only patients with measurable disease at baseline. ORR was formally compared between the two treatment arms using the Mantel-Haenszel Chi-squared test, using the randomization stratification factors. Fisher's exact test was also performed. Clinical benefit rate (CBR) was defined as the proportion of patients with a complete or partial response or with stable disease at Week 24. Exploratory analyses of CBR and time to response are provided here. Summary statistics of time to response at Week 9 will be presented as will the final analysis of the secondary endpoint of overall survival.ResultsAnalyses of ORR and CBR are provided in the following table. CapeTaxaneAnth PL (n=206)B (n=409)PL (n=104)B (n=203)PL (n=103)B (n=212)Patients with measurable disease, n (%)161 (78.2)325 (61.1)85 (81.7)161 (79.3)92 (89.3)184 (86.8)ORR, %23.635.435.350.340.252.2CR, n (%)1 (0.6)7 (2.2)3 (3.5)4 (2.5)2 (2.2)3 (1.6)PR, n (%)37 (23.0)108 (33.2)27 (31.8)77 (47.8)35 (38.0)93 (50.5)Difference between arms, %11.815.012.0p-value, unstratified0.00940.030.073Duration of ORMedian, mo7.29.28.68.46.08.1HR (95% CI)0.61 (0.39–0.96)0.75 (0.45–1.27)0.53 (0.34–0.83)Log-rank p-value0.03260.2840.0047Clinical benefit rate*, %47.164.365.471.969.980.7p-value, unstratified&lt;0.00010.240.04CR+PR at Week 9, %20.229.438.354.544.451.1B=bevacizumab, cape=capecitabine, CI=confidence interval, HR=hazard ratio, OR=objective response, PL=placebo, T/Anth=taxane/anthracycline.*For randomized patients.ConclusionsIn patients with HER2-negative MBC, the addition of bevacizumab to capecitabine, taxane or anthracycline chemotherapy induces more responses and those responses occur more rapidly. For capecitabine and anthracycline the addition of bevacizumab leads to objective responses that last longer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6084.

Abstract Background: MA17 showed that adjuvant letrozole after 5 years of tamoxifen markedly reduced the risk of recurrence in women with ER+ early stage breast cancer and improved overall survival in women presenting with node +ve disease. Most trials of early adjuvant aromatase inhibitor therapy required women to be postmenopausal at diagnosis to be eligible. We report here on a subset of women in the MA17 trial who were premenopausal at initial diagnosis and in whom subsequent menopause, prior to randomization, may have influenced their outcome on extended adjuvant letrozole.Methods: Women randomized to MA17 were divided into 2 groups: 1) pre-menopausal (&lt; 50 years of age but underwent bilateral oophorectomy at time when tamoxifen treatment started or &lt; 50 years of age at the start of tamoxifen treatment but became amenorrheic during adjuvant chemotherapy or tamoxifen treatment) and 2) post-menopausal. Disease-free survival (DFS) from time of randomization for women in these two groups were summarized by Kaplan-Meier curves and compared based on log-rank test and Cox model.Results: 889 women were identified as premenopausal and 4,277 as postmenopausal. Treatment, age, race, duration of tamoxifen, prior adjuvant chemotherapy, and mastectomy were independently associated with menopausal status on multivariate analysis. The interaction between treatment and menopausal status was statistically significant for DFS (p=0.02), indicating that women diagnosed with pre-menopausal breast cancer had significantly greater benefit (Hazard ratio=0.25, 95% CI: 0.12-0.51) with letrozole treatment in terms of DFS than those with post-menopausal status (hazard ratio=0.69 with 95% CI from 0.52 to 0.91). In comparison with women treated with placebo, pre-menopausal women on letrozole had a significantly higher incidence of arthralgia (24 vs. 16%; p=0.004) but a lower incidence of vaginal bleeding (10 vs. 16%; p=0.01). Compared with placebo post-menopausal women receiving letrozole had a higher incidence of hot flashes/flashes (55 vs. 50%; p=0.001), arthralgia (25 vs. 21%; p=0.002), myalgia (15 vs. 12%; p=0.007) and alopecia (5 vs. 3%; p=0.003).Conclusions: Letrozole after tamoxifen was more effective in improving DFS in women who were premenopausal at primary diagnosis than those who were postmenopausal. Letrozole was well tolerated in premenopausal women. These data indicate that women who are premenopausal at the time of diagnosis but become postmenopausal anytime before, or during, adjuvant tamoxifen should be considered for extended adjuvant therapy with letrozole. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 13.

Abstract Abstract #4033 Background: Aromatase activity within the breast is a source of estrogen that may cause breast cancer. Mammographic density (MD) is a risk factor for breast cancer whose biologic basis is unknown. Our study compared aromatase expression in tissue from dense and non-dense areas of the breasts of healthy volunteers.&#x2028; Methods: Participants were 40+ yrs, had a screening mammogram with visible MD, no history of cancer and were not on endocrine therapy. Ultrasound-guided core biopsies were done within 6 months of mammography to obtain three paired cores from mammographically dense and non-dense regions of the breast. Immunostaining for aromatase expression employed the streptavidin-biotin amplification method using the recently developed 677 mouse monoclonal antibody. Immunoreactivity (IR) was scored in terms of proportion of cells staining positive for aromatase (PPC) (0=&lt;1%, 1=1-25%, 2=26-50%, 3=51+% (and 4=75%+ for adipocytes)) and relative immunointensity (0=none, 1=weak, 2=moderate, 3=intense) for each cell type (stroma, normal ductal epithelium, adipocytes). A composite score weighting the PPC by their relative intensity (range 0-9; 0-12 for adipocytes) was also calculated. The sum of the composite score across the three cell types provided a global assessment of aromatase IR. Repeated measures analyses evaluated differences in aromatase IR for dense compared to non-dense tissue within and across cell types. Parameter estimates (β) indicated the average difference between dense and non-dense IR.&#x2028; Results: 18 (37%) of the 49 participants were premenopausal (median age 46 yrs). Summing across cell types, the global composite score showed increased aromatase IR on sections sampled from dense vs. non-dense regions (β=5.3,p&lt;0.001). This global difference reflected increased aromatase IR in the stroma and normal ductal epithelium from dense sections (vs. non-dense). For 42 women with &gt;1% stromal cells on each section, there was evidence for increased IR on dense sections as indicated by the greater PPCs (β=0.9), relative intensity (β=0.7) and composite score (β=2.7)(p's&lt;0.001). Similarly, for 20 women with &gt;1% normal ductal epithelium in both dense and non-dense sections, there was a greater composite score (β=1.4,p=0.004) for cells on dense sections although differences for PPC (β=0.3,p=0.11) and relative intensity (β=0.3,p=0.09) did not reach statistical significance. No differences were seen in IR for adipocytes from the two density regions (46 women). Findings were unchanged with adjustment for covariates, including proportions of each cell type per section.&#x2028; Conclusions: There is strong evidence for increased aromatase expression in the stromal and normal ductal epithelium of dense vs. non-dense tissue. These results support ongoing research into mammographic density as a biomarker of effect of aromatase inhibitors. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4033.

Luteinizing hormone (LH) administered in pharmacological amounts downregulates Leydig cell steroidogenesis. Whether reversible downregulation of physiological gonadotropin drive operates in vivo is unknown. Most of the analytical models of dose-response functions that have been constructed are biased by the assumption that no downregulation exists. The present study employs a new analytical platform to quantify potential (but not required) pulsatile cycles of LH-testosterone (T) dose-response stimulation, desensitization, and recovery (pulse-by-pulse hysteresis) in 26 healthy men sampled every 10 min for 24 h. A sensitivity-downregulation hysteresis construct predicted marked hysteresis with a median time delay to LH dose-response inflection within individual T pulses of 23 min and with median T pulse onset and recovery LH sensitivities of 1.1 and 0.10 slope unit, respectively ( P < 0.001). A potency-downregulation model yielded median estimates of one-half maximally stimulatory LH concentrations (EC50 values) of 0.66 and 7.5 IU/l for onset and recovery, respectively ( P < 0.001). An efficacy-downregulation formulation of hysteresis forecasts median LH efficacies of 20 and 8.3 ng·dl−1·min−1 for onset and offset of T secretory burst, respectively ( P = 0.002). Segmentation of the LH-T data by age suggested greater sensitivity, higher EC50 (increased LH potency), and markedly (2.7-fold) attenuated LH efficacy in older individuals. Each of the three hysteresis models yielded a marked ( P < 0.005) reduction in estimated model residual error compared with no hysteresis. In summary, model-based analyses allowing for (but not requiring) reversible pituitary-gonadal effector-response downregulation are consistent with a hypothesis of recurrent, brief cycles of LH-dependent stimulation, desensitization, and recovery of pulsatile T secretion in vivo and an age-associated reduction of LH efficacy. Prospective studies would be required to prove this aging effect.