Khaled A. Hassan

Activation of an IL6 Inflammatory Loop Mediates Trastuzumab Resistance in HER2+ Breast Cancer by Expanding the Cancer Stem Cell Population

Although inactivation of the PTEN gene has been implicated in the development of resistance to the HER2 targeting antibody trastuzumab, the mechanisms mediating this resistance remain elusive. We generated trastuzumab resistant cells by knocking down PTEN expression in HER2 overexpressing breast cancer cell lines and demonstrate that development of trastuzumab resistance in these cells is mediated by activation of an IL6 inflammatory feedback loop leading to expansion of the cancer stem cell (CSC) population. Long term trastuzumab treatment generates highly enriched CSCs which display an EMT phenotype secreting over 100-fold more IL6 than parental cells. An IL6 receptor antibody interrupted this inflammatory feedback loop reducing the cancer stem cell population resulting in decreased tumor growth and metastasis in mouse xenographs. These studies demonstrate that trastuzumab resistance may be mediated by an IL6 inflammatory loop and suggest that blocking this loop may provide alternative strategy to overcome trastuzumab resistance.

Notch pathway activity identifies cells with cancer stem cell-like properties and correlates with worse survival in lung adenocarcinoma

PURPOSE The cancer stem cell theory postulates that tumors contain a subset of cells with stem cell properties of self-renewal, differentiation, and tumor initiation. The purpose of this study is to determine the role of Notch activity in identifying lung cancer stem cells. EXPERIMENTAL DESIGN We investigated the role of Notch activity in lung adenocarcinoma using a Notch GFP reporter construct and a γ-secretase inhibitor (GSI), which inhibits Notch pathway activity. RESULTS Transduction of lung cancer cells with Notch GFP reporter construct identified a subset of cells with high Notch activity (GFP-bright). GFP-bright cells had the ability to form more tumor spheres in serum-free media and were able to generate both GFP-bright and GFP-dim (lower Notch activity) cell populations. GFP-bright cells were resistant to chemotherapy and were tumorigenic in serial xenotransplantation assays. Tumor xenografts of mice treated with GSI had decreased expression of downstream effectors of Notch pathway and failed to regenerate tumors upon reimplantation in NOD/SCID mice. Using multivariate analysis, we detected a statistically significant correlation between poor clinical outcome and Notch activity (reflected in increased Notch ligand expression or decreased expression of the negative modulators), in a group of 443 patients with lung adenocarcinoma. This correlation was further confirmed in an independent group of 89 patients with adenocarcinoma in which Hes-1 overexpression correlated with poor overall survival. CONCLUSIONS Notch activity can identify lung cancer stem cell-like population and its inhibition may be an appropriate target for treating lung adenocarcinoma.

An Embryonic Stem Cell–Like Signature Identifies Poorly Differentiated Lung Adenocarcinoma but not Squamous Cell Carcinoma

Purpose: An embryonic stem cell (ESC) profile correlates with poorly differentiated breast, bladder, and glioma cancers. In this article, we assess the correlation between the ESC profile and clinical variables in lung cancer. Experimental Design: Microarray gene expression analysis was done using Affymetrix Human Genome U133A on 443 samples of human lung adenocarcinoma and 130 samples of squamous cell carcinoma (SCC). To identify gene set enrichment patterns, we used the Genomica software. Results: Our analysis showed that an increased expression of the ESC gene set and a decreased expression of the Polycomb target gene set identified poorly differentiated lung adenocarcinoma. In addition, this gene expression signature was associated with markers of poor prognosis and worse overall survival in lung adenocarcinoma. However, there was no correlation between this ESC gene signature and any histologic or clinical variable assessed in lung SCC. Conclusions: This work suggests that not all poorly differentiated non–small cell lung cancers exhibit a gene expression profile similar to that of ESC, and that other characteristics may play a more important role in the determination of differentiation and survival in SCC of the lung. (Clin Cancer Res 2009;15(20):6386–90)

Notch Reporter Activity in Breast Cancer Cell Lines Identifies a Subset of Cells with Stem Cell Activity

Developmental pathways such as Notch play a pivotal role in tissue-specific stem cell self-renewal as well as in tumor development. However, the role of Notch signaling in breast cancer stem cells (CSC) remains to be determined. We utilized a lentiviral Notch reporter system to identify a subset of cells with a higher Notch activity (Notch+) or reduced activity (Notch−) in multiple breast cancer cell lines. Using in vitro and mouse xenotransplantation assays, we investigated the role of the Notch pathway in breast CSC regulation. Breast cancer cells with increased Notch activity displayed increased sphere formation as well as expression of breast CSC markers. Interestingly Notch+ cells displayed higher Notch4 expression in both basal and luminal breast cancer cell lines. Moreover, Notch+ cells demonstrated tumor initiation capacity at serial dilutions in mouse xenografts, whereas Notch− cells failed to generate tumors. γ-Secretase inhibitor (GSI), a Notch blocker but not a chemotherapeutic agent, effectively targets these Notch+ cells in vitro and in mouse xenografts. Furthermore, elevated Notch4 and Hey1 expression in primary patient samples correlated with poor patient survival. Our study revealed a molecular mechanism for the role of Notch-mediated regulation of breast CSCs and provided a compelling rationale for CSC-targeted therapeutics. Mol Cancer Ther; 14(3); 779–87. ©2015 AACR.

Effect of Midtreatment PET/CT-Adapted Radiation Therapy With Concurrent Chemotherapy in Patients With Locally Advanced Non–Small-Cell Lung Cancer: A Phase 2 Clinical Trial

Importance Our previous studies demonstrated that tumors significantly decrease in size and metabolic activity after delivery of 45 Gy of fractionated radiatiotherapy (RT), and that metabolic shrinkage is greater than anatomic shrinkage. This study aimed to determine whether 18F-fludeoxyglucose–positron emission tomography/computed tomography (FDG-PET/CT) acquired during the course of treatment provides an opportunity to deliver higher-dose radiation to the more aggressive areas of the tumor to improve local tumor control without increasing RT-induced lung toxicity (RILT), and possibly improve survival. Objective To determine whether adaptive RT can target high-dose radiation to the FDG-avid tumor on midtreatment FDG-PET to improve local tumor control of locally advanced non–small-cell lung cancer (NSCLC). Design, Setting, and Participants A phase 2 clinical trial conducted at 2 academic medical centers with 42 patients who had inoperable or unresectable stage II to stage III NSCLC enrolled from November 2008, to May 2012. Patients with poor performance, more than 10% weight loss, poor lung function, and/or oxygen dependence were included, providing that the patients could tolerate the procedures of PET scanning and RT. Intervention Conformal RT was individualized to a fixed risk of RILT (grade >2) and adaptively escalated to the residual tumor defined on midtreatment FDG-PET up to a total dose of 86 Gy in 30 daily fractions. Medically fit patients received concurrent weekly carboplatin plus paclitaxel followed by 3 cycles of consolidation. Main Outcomes and Measures The primary end point was local tumor control. The trial was designed to achieve a 20% improvement in 2-year control from 34% of our prior clinical trial experience with 63 to 69 Gy in a similar patient population. Results The trial reached its accrual goal of 42 patients: median age, 63 years (range, 45-83 years); male, 28 (67%); smoker or former smoker, 39 (93%); stage III, 38 (90%). Median tumor dose delivered was 83 Gy (range, 63-86 Gy) in 30 daily fractions. Median follow-up for surviving patients was 47 months. The 2-year rates of infield and overall local regional tumor controls (ie, including isolated nodal failure) were 82% (95% CI, 62%-92%) and 62% (95% CI, 43%-77%), respectively. Median overall survival was 25 months (95% CI, 12-32 months). The 2-year and 5-year overall survival rates were 52% (95% CI, 36%-66%) and 30% (95% CI, 16%-45%), respectively. Conclusions and Relevance Adapting RT-escalated radiation dose to the FDG-avid tumor detected by midtreatment PET provided a favorable local-regional tumor control. The RTOG 1106 trial is an ongoing clinical trial to validate this finding in a randomized fashion. Trial Registration clinicaltrials.gov Identifier: NCT01190527

Lung cancer stem cell: Fancy conceptual model of tumor biology or cornerstone of a forthcoming therapeutic breakthrough?

Cancer research has received a fresh impetus from the concept of cancer stem cell (CSC) which postulates the existence of a tumor cell population uniquely endowed with self-renewal capacity and therapy resistance. Despite recent progresses including targeted therapy, lung cancer treatment remains a challenge owing largely to disease recurrence. Providing a conceptual model of tumor resistance and disease relapse, the lung CSC has received extensive attention, leading to a flourishing literature and several ongoing clinical trials. In this study, we will discuss the data suggesting the existence of CSC in lung tumors and the potential clinical utility of CSCs as prognostic markers or cellular targets of new therapeutic strategies. We will also touch on the new fundamental developments of the CSC concept that ought to be considered if the integration of the CSC concept into clinical practice is to be successful and impact on lung cancer treatment.

Long-term survival in paraneoplastic opsoclonus-myoclonus syndrome associated with small cell lung cancer

Paraneoplastic opsoclonus-myoclonus syndrome (OMS) is associated with small cell lung cancer (SCLC) in adults. Without appropriate treatment for SCLC, all reported patients with SCLC and OMS have died of complications of OMS within 3 months of diagnosis. With appropriate treatment, about half of reported patients have had improvement in neurologic function, and several have become long-term survivors (6-84 months). We report a patient with SCLC who presented with OMS and was refractory to immunosuppressive therapy but responded rapidly to antineoplastic therapy and remains alive with no sign of SCLC recurrence and minimal residual neurologic deficits 30 months after diagnosis. In patients presenting with OMS, early recognition and treatment of the underlying malignancy probably improve the chances for recovery from the OMS with minimal deficit and ultimate survival.

Abstract 4119: The development of EGFR resistant mutation, T790M, in lung adenocarcinoma is acquired through a specific cytosine deamination mechanism

EGFR resistant mutation, T790M, in lung adenocarcinoma is acquired through a specific cytosine deamination mechanism. Background: Epidermal growth factor receptor (EGFR) activation mutations occur in 15% of lung adenocarcinomas. This leads to constitutive activation of EGFR, which triggers multiple downstream survival and proliferation pathways. Currently, EGFR tyrosine kinase inhibitors (TKIs) are first line therapy for stage IV non-small cell lung cancer (NSCLC) patients with EGFR mutations. Despite initial significant response to TKIs, most tumors develop resistance. The main mechanism of resistance detected in 50-60% of cases is a cytosine to thymine (C>T) single nucleotide transition mutation at position 2369. This causes a threonine to methionine amino acid change at position 790 (i.e. T790M). Our data suggests that the C>T mutation is an acquired event secondary to cytosine deamination by Activation Induced Cytosine Deamination enzyme (AICDA). Results: Single cell clones of lung adenocarcinoma cell line, PC9, were treated with EGFR TKI. At baseline, these clones have EGFR exon 19 deletion but no evidence of T790M mutation by digital droplet PCR (ddPCR). However, after treatment with a serial increasing dose of EGFR TKI, T790M mutation was detected by ddPCR. Assessing whether cytosine deamination enzymes were altered by this treatment, a significant increase in AICDA expression was seen. Furthermore, recombinant AICDA protein could deaminate cytosine at position 2369 in vitro. In addition, using mass spectrometry and methylation specific primers, we determined that cytosine at position 2369 is in fact methylated. This further supports our hypothesis since 5-methyl cytosine is deaminated into thymine directly. Since in germinal center B-lymphocytes, AICDA is activated through a non-canonical NFkB mechanism, we assessed NFkB pathway in PC9 cell line. RelB and p52 expression were significantly increased after TKI treatment. In addition direct interaction between RelB and AICDA promoter was confirmed by ChIP Assay. These findings were also seen in a mouse PC9 xenograft model. Daily oral gavage of EGFR TKI caused significant increase in the expression of RelB as well as AICDA. Adding NFkB inhibitor twice weekly inhibited the expression of RelB and AICDA. Finally, knocking down AICDA by shRNA prevented the development of T790M mutation in PC9 cell lines after TKI exposure. Conclusion: Our data suggest that the T790M mutation could be actively acquired after TKI treatment through a cytosine deamination process by AICDA. This would have significant implications for treatment with targeted therapy. In fact, Imatinib resistance in CML and GIST tumors have a similar C>T single nucleotide transition mutation. Citation Format: Khaled Hassan, Najwa El Kadi, April Davis, Gregory Kalemkerian, Luo Wang, Hasan Korkaya. The development of EGFR resistant mutation, T790M, in lung adenocarcinoma is acquired through a specific cytosine deamination mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4119. doi:10.1158/1538-7445.AM2017-4119

Abstract 739: Tumor cell plasticity with transition to a mesenchymal phenotype is a mechanism of chemoresistance that is reversed by Notch pathway inhibition in lung adenocarcinoma

Background: Lung cancer is the leading cause of cancer death in the world, mainly due to complex genetic events that characterize lung cancer, tumor heterogeneity and resistance to traditional and targeted treatments. Resistance may be explained by passive clonal selectivity, however, the propensity and variability of resistance suggests an active process of tumor plasticity. We hypothesize that tumor cells undergo an epithelial to mesenchymal transition as a mechanism of resistance to chemotherapy. Results: The lung adenocarcinoma cell lines NCI-H1299, NCI-H358, and NCI-H441 were exposed to gradually increasing doses of docetaxel over 12-16 weeks. Chemoresistance emerged with a 10-15X increase in IC50 as compared to parental cells. With each treatment dose increase more than 50% of cells remained alive, suggesting bulk cell transformation. This resistance has been maintained for more than 20 generations after withdrawal of docetaxel. Morphologically, NCI-H358 and NCI-H441 chemoresistant cell lines lost their cobblestone architecture becoming more spindle-shaped. Using RT-PCR assays, there was increased expression of mesenchymal-related genes, vimentin (2.5-3.5X), twist (2-4X) and decreased expression of epithelial-related genes such as E-CAD (2-5X). Our previous data suggested that Notch activity plays a significant role in the EMT process, thus we evaluated Notch downstream genes expression by RT-PCR. We detected increase expression of Hey2 (2.5-3.5X), in NCI-H358 and NCI-H441 resistant cell lines as compared to parental cells. When chemoresistant cell lines were exposed to gamma secretase inhibitor, a Notch inhibitor, they became less invasive, epithelial-like and sensitive to docetaxel. Conclusion: These data suggest that tumor cell plasticity with transition to a mesenchymal phenotype is a mechanism of chemotherapy resistance that is controlled through the Notch pathway. Inhibiting Notch activity induces mesenchymal to epithelial transition and re-sensitizes cells to chemotherapy. Notch inhibition in combination with standard chemotherapy represents a potential approach to achieve better disease control. Citation Format: Khaled A. Hassan. Tumor cell plasticity with transition to a mesenchymal phenotype is a mechanism of chemoresistance that is reversed by Notch pathway inhibition in lung adenocarcinoma. [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 739. doi:10.1158/1538-7445.AM2015-739

Abstract 3349: Transformation of mammary epithelial cells by inactivation of p53 and PTEN generates cancer stem cells driven by an IL-6/TGF-beta inflammatory loop

The PTEN and p53 tumor suppressor genes, frequently inactivated in human breast cancers have been shown to be regulators of normal and malignant breast stem cell self-renewal. In addition, inactivation of tumor suppressors in preneoplastic lesions are implicated in tumor progression from ductal situ in carcinoma (DCIS) to invasive ductal carcinoma. To determine the relationship between stem cell self-renewal and carcinogenesis, we examined the effects of PTEN and p53 knockdown on normal mammary epithelial cells obtained from reduction mammoplasties, as well as in the non-transformed mammary cell line MCF10A. Simultaneous knockdown of PTEN and p53 in normal mammary epithelial cells generated hyperplastic and DCIS-like lesions when these cells were transplanted into the humanized mammary fat pads of NOD/SCID mice. In MCF10A cells knockdown of PTEN and p53 synergized to expand the stem cell compartment transforming these cells and generating highly metastatic tumors in NOD/SCID mice. Stem cell expansion was driven by activation of an inflammatory loop involving both IL-6 and TGF-b. This inflammatory loop was mediated by both AKT/b-Catenin and Stat3/NF-kB pathways. Conversely collective inhibition of these pathways including TGF-b, IL-6 and Wnt/b-Catenin and Stat3/NF-kB was necessary to revert back this stem cell phenotype. This may suggest that synergistic activity of these pathways may contribute to the expansion and transformation of mammary stem cells. The expanded cancer stem cells were highly metastatic and invasive and displayed properties of EMT. The IL6-R antibody tocilizumab blocked this loop resulting in a reduction of CSCs and thus inhibiting tumor growth in mouse xenografts. These studies suggest that loss of PTEN and p53 transforms mammary epithelial cells by activating an inflammatory loop mediated by IL-6 and TGF-b, generating CSCs with an EMT phenotype. Since CSCs drive tumor growth and metastasis, targeting the self-renewal by interfering with this CSC, inflammatory loop represents a rational strategy for breast cancer prevention and therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3349. doi:1538-7445.AM2012-3349