Aiwu R. He

Evaluation of Hypertension as a Marker of Bevacizumab Efficacy

BackgroundPredictive factors for efficacy of vascular endothelial growth factor pathway-targeted therapies have not been identified or confirmed. Hypertension has been observed as a side effect to anti-vascular endothelial growth factor therapy. The goal of our study was to retrospectively assess if hypertension induced during treatment with bevacizumab was associated with clinical outcome in metastatic colorectal cancer patients treated with bevacizumab.Patients and methodsWe conducted a retrospective chart review of patients with colorectal cancer treated with bevacizumab at Lombardi Comprehensive Cancer Center from 2004 to 2008.ResultsEighty-four patients with metastatic colorectal cancer were eligible. Eighteen patients (21%) developed grades 3 hypertension. Twelve patients (14%) developed grade 2 hypertension. Six patients (7%) developed grade 1 hypertension. Median overall survival (OS) was 29xa0months and progression-free survival (PFS) was 10xa0months. Patients with any grade hypertension while on bevacizumab had an adjusted hazard ratio for death of 0.32 (pu2009=u20090.03) and adjusted risk of progression of 51% (pu2009=u20090.02) compared to those without hypertension (HTN). When stratified by metastatic disease, patients presenting with metastases who developed HTN had better OS and PFS (pu2009=u20090.03 and 0.01.) Among patients without metastases at diagnosis, those with HTN on bevacizumab had better OS and PFS but results were not statistically significant (pu2009=u20090.60 and 0.62, respectively).ConclusionsOur data indicate that bevacizumab-induced hypertension may represent an interesting prognostic factor for clinical outcome in advanced colorectal cancer patients receiving bevacizumab.

βII-Spectrin (SPTBN1) suppresses progression of hepatocellular carcinoma and Wnt signaling by regulation of Wnt inhibitor kallistatin.

βII‐Spectrin (SPTBN1) is an adapter protein for Smad3/Smad4 complex formation during transforming growth factor beta (TGF‐β) signal transduction. Forty percent of SPTBN1+/− mice spontaneously develop hepatocellular carcinoma (HCC), and most cases of human HCC have significant reductions in SPTBN1 expression. In this study, we investigated the possible mechanisms by which loss of SPTBN1 may contribute to tumorigenesis. Livers of SPTBN1+/− mice, compared to wild‐type mouse livers, display a significant increase in epithelial cell adhesion molecule‐positive (EpCAM+) cells and overall EpCAM expression. Inhibition of SPTBN1 in human HCC cell lines increased the expression of stem cell markers EpCAM, Claudin7, and Oct4, as well as decreased E‐cadherin expression and increased expression of vimentin and c‐Myc, suggesting reversion of these cells to a less differentiated state. HCC cells with decreased SPTBN1 also demonstrate increased sphere formation, xenograft tumor development, and invasion. Here we investigate possible mechanisms by which SPTBN1 may influence the stem cell traits and aggressive behavior of HCC cell lines. We found that HCC cells with decreased SPTBN1 express much less of the Wnt inhibitor kallistatin and exhibit decreased β‐catenin phosphorylation and increased β‐catenin nuclear localization, indicating Wnt signaling activation. Restoration of kallistatin expression in these cells reversed the observed Wnt activation. Conclusion: SPTBN1 expression in human HCC tissues is positively correlated with E‐cadherin and kallistatin levels, and decreased SPTBN1 and kallistatin gene expression is associated with decreased relapse‐free survival. Our data suggest that loss of SPTBN1 activates Wnt signaling, which promotes acquisition of stem cell‐like features, and ultimately contributes to malignant tumor progression. (Hepatology 2015;61:598‐612)

Irreversible Multitargeted ErbB Family Inhibitors for Therapy of Lung and Breast Cancer

Overactivation of the ErbB protein family, which is comprised of 4 receptor tyrosine kinase members (ErbB1/epidermal growth factor receptor [EGFR]/HER1, ErbB2/HER2, ErbB3/HER3, and ErbB4/HER4), can drive the development and progression of a wide variety of malignancies, including colorectal, head and neck, and certain non-small cell lung cancers (NSCLCs). As a result, agents that target a specific member of the ErbB family have been developed for the treatment of cancer. These agents include the reversible EGFR tyrosine kinase inhibitors (TKIs) erlotinib and gefitinib; the EGFR-targeting monoclonal antibodies cetuximab and panitumumab; and the HER2-targeting monoclonal antibody trastuzumab. Lapatinib is a dual TKI that targets both EGFR and HER2. In addition, TKIs that inhibit multiple members of the ErbB family and also bind their targets irreversibly are under evaluation for the treatment of cancer. Three such compounds have progressed into clinical studies: the EGFR, HER2, and HER4 inhibitors afatinib, dacomitinib, and neratinib. Phase I studies of these agents have shown clinical activity in NSCLC, breast cancer, and other malignancies. Currently, afatinib is approved for EGFR mutation-positive NSCLC and is in development for squamous NSCLC, and dacomitinib is in phase III of clinical development for NSCLC, neratinib is in phase III of clinical development for the treatment of breast cancer, and afatinib is also in phase III development in head and neck cancer. Final results from clinical trials may lead to the potential approval of these agents in a variety of solid tumor malignancies.

Phase I trial of ixabepilone administered as three oral doses each separated by 6 hours every 3 weeks in patients with advanced solid tumors.

SummaryBackground Ixabepilone, which stabilizes microtubules, has low susceptibility to drug resistance mediated by P-glycoprotein or βIII-tubulin. Materials and Methods This study was designed to determine the maximum tolerated dose (MTD) of oral ixabepilone when administered every 6xa0h for three doses, every 3xa0weeks, to patients with refractory advanced cancers. Eighteen patients were treated with escalating doses of ixabepilone: three at cohort 1 (30xa0mg/dose; 90xa0mg on Day 1), nine at cohort 2 (40xa0mg/dose; 120xa0mg on Day 1), and six at cohort 3 (50xa0mg/dose; 150xa0mg on Day 1). Serial plasma samples were collected during cycle 1 for pharmacokinetic (PK) measurements. Results Of the 18 treated patients, eight were male and ten were female. The median age was 59xa0years, and most had an excellent performance status (KPS 90–100; 61%). There were two dose limiting toxicities (DLT): Grade 4 febrile neutropenia at the 120xa0mg dose and Grade 4 neutropenic sepsis at the 150xa0mg dose. Because of the severity and duration of neutropenic sepsis at level 3, level 2 (120xa0mg) was defined as the MTD and this cohort was expanded to nine patients. High inter-individual variability in plasma drug concentrations was observed during the study, with particularly high levels in two patients with DLT. Conclusions On the basis of this safety profile, the MTD of oral ixabepilone was defined as 120xa0mg given as three 40xa0mg doses each separated by 6xa0h on Day 1 of a 3-week cycle. However, the PK variability observed makes further development of this oral formulation unlikely.

Phase I Study of Single-Agent Utomilumab (PF-05082566), a 4-1BB/CD137 Agonist, in Patients with Advanced Cancer

Purpose: Utomilumab (PF-05082566) is an agonistic mAb that engages the immune costimulatory molecule 4-1BB/CD137. In this first-in-human, phase I, open-label, multicenter, multiple-dose study (NCT01307267) we evaluated safety, tolerability, pharmacokinetics, preliminary clinical activity, and pharmacodynamics of single-agent utomilumab in patients with advanced malignancies. Experimental Design: Dose escalation was based on a standard 3+3 design for doses of utomilumab from 0.006 to 0.3 mg/kg every 4 weeks and a time-to-event continual reassessment method for utomilumab 0.6 to 10 mg/kg every 4 weeks. The primary study endpoint was dose-limiting toxicity (DLT) in the first two cycles. Results: Utomilumab demonstrated a well-tolerated safety profile (N = 55). None of the patients experienced a DLT at the dose levels evaluated. The most common treatment-related adverse events were fatigue, pyrexia, decreased appetite, dizziness, and rash (<10% of patients). Only one (1.8%) patient experienced a grade 3–4 treatment-related adverse event (fatigue), and no clinically relevant elevations in transaminases were noted. Utomilumab demonstrated linear pharmacokinetics at doses ranging from 0.006 to 10 mg/kg, with similar safety and pharmacokinetics in anti-drug antibody (ADA)-negative and ADA-positive patients. The overall objective response rate was 3.8% (95% CI, 0.5%–13.0%) in patients with solid tumors and 13.3% in patients with Merkel cell carcinoma, including a complete response and a partial response. Circulating biomarkers support 4-1BB/CD137 engagement by utomilumab and suggest that circulating lymphocyte levels may influence probability of clinical benefit. Conclusions: The favorable safety profile and preliminary antitumor activity demonstrated by utomilumab warrant further evaluation in patients with advanced malignancies. Clin Cancer Res; 24(8); 1816–23. ©2018 AACR.

Abstract 892: Vitamin D deficiency regulates TLR7 to promote hepatocellular cancer in TGF-β/Smad3 heterozygous mice

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PAnnHepatocellular carcinoma (HCC) is the fifth most common tumor and the third leading cause of cancer-related deaths. Loss of TGF-β signaling has been associated with development of HCC. Cancer preventive effects of Vitamin D (VD) have been implicated in multiple cancers, however a clear role for Vitamin D in specific high risk populations remains undefined for HCC. Therefore, we examined for a potential chemopreventive role of VD in HCC in the context of TGF-β inactivation.nnMethods: (1) We screened for somatic mutation of the TGF-β pathway and VD related genes in 202 HCCs from The Cancer Genome Atlas (TCGA). (2) Wild type, Sptbn1+/- and Smad3+/- mice were fed with low VD (200 IU VD/kg) or high VD diet (10,000 IU VD/kg) for 9 weeks. Liver tissues were subjected to microarray analyses and further evaluation by quantitative PCR. (3) Wild type, Sptbn1+/- and Smad3+/- mice were injected Diethylnitrosamine (DEN) and at 8 month, 26 mice receiving low VD (200 IU VD/kg) and 26 mice receiving high VD (10,000 IU VD/kg) chow. HCC development was assessed at 4 months after VD treatment. (4) Reverse Phase Protein Array (RPPA) was performed to analyze expression profiles of 164 proteins of mouse liver tumors. (5) Liver samples from patients with HCV cirrhosis receiving VD supplements were examined to evaluate expression of TGF-β, Wnt and VD pathway molecules by immunohistochemistry.nnResults: (1) We observed a high rate of somatic mutation in TGF-β and VD pathway related genes in the TCGA genomic analysis. (2) None of the VD treated mice developed HCC but high VD treatment increased TLR7 mRNA expression about 3-fold in liver from Smad3+/- mice compared with livers from WT mice. (3) Smad3+/- mice with low VD showed 3-fold larger HCC formation, compared to the high VD group (Smad3+/-) that did not develop significant tumors. However, correction of VD in the Chow after 10 months did not reverse HCC formation. (4) RPPA data revealed that the tumor suppressor protein PDCD4 was reduced in Smad3+/- mice with low VD treatment. However, oncoproteins such as β-catenin, Stat5A and Bcl2-XL were induced in the same sample. (5) Expression of β2SP and TβRII were higher in HCV cirrhosis patients receiving VD supplementation compared to non-treated group.nnConclusions: Loss of TGF-β signaling pathway developed HCC and VD deficiency promotes tumor growth in the context of Smad3 disruption potentially through regulation of TLR7 expression. However, after 10 months restoring VD does not have any significant effect on altering tumors. Therefore VD could be a potential candidate for prevention in early identified HCC high risk individuals who has inactivation of TGF-β/Smad3 signaling.nnCitation Format: Ji-hyun Shin, Lior H. Katz, Nina M. Munoz, Andrea Cortes, Vivek Shukla, Sang-Bae Kim, Franklin Herlong, Keigo Machida, Hidekazu Tsukamoto, Kirti Shetty, Aiwu R. He, Lynt B. Johnson, Asif Rashid, Jian Chen, Ju-Seog Lee, Lopa Mishra. Vitamin D deficiency regulates TLR7 to promote hepatocellular cancer in TGF-β/Smad3 heterozygous mice. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 892. doi:10.1158/1538-7445.AM2015-892

Abstract 5275: βII-spectrin(SPTBN1) suppresses tumor progression by inhibiting Wnt signaling via kallistatin in hepatocellular carcinoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnβII-spectrin(SPTBN1, β2SP), the most common nonerythrocytic member of the β-spectrin gene family, functions as an adapter protein for Smad3/Smad4 complex formation during TGF-β signal transduction, and is required for EMT during embryonic liver development. Moreover, β2SP is emerging as a tumor suppressor. We have reported that Loss of β2SP enhances the stem-like traits and invasiveness of HCC cells. The canonical Wnt pathway, known to be a critical regulator of self-renewal in stem cell, is also constitutively activated and implicated in the induction of EMT in cancer. Kallistatin (a serine protease inhibitor, SERPINA4), which was identified as a unique inhibitor of the Wnt pathway, is expressed primly in the liver. In this study, we investigated the mechanism by which β2SP influences stem cell traits and aggressive behavior of HCC cell lines. We found that cells with decreased β2SP have decreased β-catenin phosphorylation and increased β-catenin nuclear localization, indicative of activation of Wnt signaling. Furthermore, loss of β2SP results in the decease of Kallistatin expression, the Wnt inhibitor. Restore of kallistatin reversed the Wnt activation in HCC cells from decreased β2SP level. Our study provides evidence for the first time that loss of β2SP activates Wnt signal through suppressing kallistatin expression.nnNote: This abstract was not presented at the meeting.nnCitation Format: Xiuling Zhi, Ling Lin, Narayan Shivapurkar Shivapurkar, John L. Marshall, Lynt Johnson, Aiwu R. He. βII-spectrin(SPTBN1) suppresses tumor progression by inhibiting Wnt signaling via kallistatin in hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5275. doi:10.1158/1538-7445.AM2014-5275

Abstract 248: Vitamin D deficiency promotes hepatocellular carcinoma tumor growth in TGF-β impaired mice by Smad3 heterozygous deletion

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related deaths worldwide. Vitamin D (VD) has been implicated in the prevention of multiple cancers, some of which are characterized by inactivation of TGF-β signaling. Recently, inactivation of TGF-β signaling has been associated with development of HCC. We examined the potential role of VD in HCC chemoprevention in the context of TGF-β inactivation in a mouse model. Methods: Wild type, Sptbn1+/- and Smad3+/- male mice were treated with 50 mg/kg of the chemical carcinogen diethylnitrosamine (DEN) via i.p. injection at 14 days of age. At 8 months, the animals were changed from regular chow (2,200 IU/kg VD) to one of two diets: lower than normal VD (200 IU/kg) or high VD (10,000 IU/kg). At one year of age, the livers were harvested for tissue analysis. Several metrics were assessed including expression levels of 164 proteins from liver tumors by Reverse Phase Protein Array (RPPA). Results: Twenty three mice were treated with lower than normal dose VD while 26 received high dose VD. We did not find significant differences in blood calcium levels in animals on either diet. As expected, high VD intervention after 8 months of DEN injection did not affect the total number of tumors the mice developed; however, the lower than normal VD regimen promoted tumor growth in the context of Smad3 disruption (liver-to-body weight ratio 18.37% in Smad3+/- mice treated with low dose VD vs. 6.25% in Smad3+/- animals treated with high dose, p=0.028). Furthermore, RPPA data revealed that the lower than normal VD intake in DEN-treated Smad3 heterozygous null mice results in repression of tumor suppressing genes such as PDCD4 and concomitant up-regulation of tumor promoting genes like Stat5A, Bcl2-XL, PDGFRB and PEA15. Conclusions: Remarkably, lower than normal VD in the context of Smad3 disruption promotes tumor growth possibly through repression of tumor suppressing genes such as PDCD4 and up-regulation of oncogenes like Stat5A, Bcl2-XL, PDGFRB and PEA15. These results suggest that VD treatment strategies could potentially be significant for chemoprevention and treatment of HCC in the context of inactivation of TGF-β signaling in patient populations with underlying deficiency of VD. Citation Format: Nina M. Munoz, Lior H. Katz, Keigo Machida, Hidekazu Tsukamoto, Kirty Shetty, Aiwu R. He, Lynt B. Johnson, Asif Rashid, Sang Bae Kim, Ju-Seog Lee, Lopa Mishra. Vitamin D deficiency promotes hepatocellular carcinoma tumor growth in TGF-β impaired mice by Smad3 heterozygous deletion. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 248. doi:10.1158/1538-7445.AM2014-248

Abstract 4111: Vitamin D for prevention of liver cancer in the setting of disrupted TGF-β signaling pathway

Hepatocellular carcinoma (HCC) is the third most common cause of cancer related death worldwide. Vitamin D (VD) has been implicated in the prevention of multiple cancers- some with inactivation of TGF-β signaling. Recently, inactivation of TGF-β signaling has been associated with HCC. We analyzed whole genome data for TGF-β signaling and examined the role of VD in the context of TGF-β inactivation in HCC. Methods: Databases of HCC Genomics (COSMIC) and transcriptomics (TCGA) were analyzed. The effect of calcitriol on cell proliferation was measured in HCC cell lines; Hep-G2 and MEF cells, with knocked down TGF-β/β2-spectrin (β2SP). Wild-type (WT), Sptbn1+/- and Smad3+/- mice were fed with diets containing 200 IU VD/kg or 10,000 IU VD/kg for 9 weeks. Hepatocyte proliferation was analyzed through Ki67 staining. The expression of cyclin D1 was evaluated by Western blotting. A whole genome gene expression profiling array was performed from liver samples of those mice using Illumina MouseWG-6 v2.0 gene expression arrays®. Lastly, liver samples from patients with HCC who had received VD supplementation were evaluated by immunohistochemistry. Results: Whole genome data revealed aberrant TGF-β signaling in ∼70% of HCCs. Treatment with calcitriol suppressed the growth of HCC cell lines, regardless of their TGF-β pathway status. Inhibition of proliferation was noted in WT MEFs as well as in the Sptbn1+/- and Sptbn1-/- cells. High dose VD reduced hepatic proliferation and cyclin D1 levels in Sptbn1+/- and Smad3+/- mutants, and in WT mice. The microarray data demonstrated the most significant changes in gene expression were associated with acute phase inflammatory response (p Citation Format: Lior H. Katz, Andrea Cortes, Vivek Shukla, Keigo Machida, Hidekazu Tsukamoto, Kirty Shetty, Aiwu R. He, Lynt B. Johnson, Jian Chen, Randa El-Zein, Ju-Seog Lee, Lopa Mshra. Vitamin D for prevention of liver cancer in the setting of disrupted TGF-β signaling pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4111. doi:10.1158/1538-7445.AM2014-4111

Abstract 3764: Loss of β2SP (ELF) enhances the stem-like traits and invasiveness of HCC cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnβ2SP(ELF) is one of adapter proteins for Smad3/Smad4 complex formation during TGF-β signal transduction. 40% of β2SP+/- mice spontaneously develop hepatocellular carcinoma (HCC) and most cases of human HCC, gastric cancer, and lung cancer demonstrate significant reductions in β2SP expression. In our study, we investigated the possible mechanism for loss of β2SP induced HCC tumorigenesis. Recent studies have highlighted the involvement of cancer stem cells during the formation and progression of various types of cancers, epithelial cells that have undergone EMT (epithelial mesenchymal transition) acquire stem-cell properties during cancer development. Giving the role of TGF-beta signaling in stem cell differentiation and EMT during embryonic development, we hypothesize loss of β2SP results in alteration of liver stem cell that may contribute to the development of HCC in β2SP+/− mice. It has been shown that EpCAM-positive adult liver cells has stem cell property and can differentiate into functional hepatocytes in xenograft model. We found that EpCAM-positive hepatocyte number doubled in β2SP+/− mouse compared to wild type by fluorescence-activated cell sorter (FACS). Furthermore EpCAM level in β2SP+/− mouse liver was three times higher than that in the liver of wild type mouse. To study the mechanism by which Epcam is regulated by β2SP, HCC cells were transfected by β2SP siRNA. Inhibition of β2SP increased the expression of Epcam, and EMT markers vimentin was increased and E-cadherin expression was decreased in PLC/PRF/5 and SNU449 cells, while TGF-β1 and Smad3 rescued the expression of Epcam and E-cadherin, but not vimentin. We also found that suppression of β2SP expression promotes adhesion, migration and cloning formation of PLC/PRF/5 and SNU449 cells. Our data suggests loss of β2SP enhances the stem-like traits and invasiveness of HCC cells.nnCitation Format: Xiuling Zhi, Ling Lin, Narayan Shivapurkar, Wilma Jogunoori, Lopa Mishra, Aiwu R He. Loss of β2SP (ELF) enhances the stem-like traits and invasiveness of HCC cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3764. doi:10.1158/1538-7445.AM2013-3764