Suzanne Meredith

Biomarkers for small cell lung cancer: neuroendocrine, epithelial and circulating tumour cells.

Small cell lung cancer (SCLC) is characterised by an aggressive clinical course with invariable resistance to chemotherapy despite initially high response rates. There has been little improvement in outcome over the past few decades, with no breakthrough yet in targeted therapies. Recent preclinical data and studies of circulating tumour cells (CTCs) highlight distinct cellular heterogeneity within SCLC. Better understanding of how these phenotypes contribute to metastasis and tumour progression might pave the way for development of more successful targeted therapies. Here we review these studies, their implications for future research and for the incorporation of biomarkers reflecting neuroendocrine, epithelial and mesenchymal phenotypes in clinical studies.

Neuroendocrine and epithelial phenotypes in small-cell lung cancer: implications for metastasis and survival in patients

Background:Small-cell lung cancer (SCLC) has a very aggressive clinical course with early metastasis. This study investigated how the distinctive neuroendocrine characteristics contribute to disease progression and invasion in human SCLC.Methods:The neuroendocrine phenotype (pro-opiomelanocortin (POMC)) was quantified by ELISA in blood samples from 43 SCLC patients. The neuroendocrine (POMC, chromogranin A, neuron-specific enolase, NCAM) and epithelial (cytokeratin and E-cadherin) phenotypes were investigated, using ELISA and immunocytochemistry/immunohistochemistry.Results:In SCLC patients, 16% had elevated circulating POMC, which was associated with significantly worse survival (P=0.02) and liver metastases (P=0.004). In addition, POMC correlated with epithelial-positive circulating tumour cells (P=0.0002). In a panel of SCLC cell lines, all POMC-secreting cell lines expressed cytokeratin (40% of total). Even after cloning, DMS 79 cells expressed both neuroendocrine and epithelial markers. DMS 79 xenografts secreted POMC into the blood, which mirrored the tumour volume. These xenografts expressed both neuroendocrine and epithelial phenotypes in all tumours, with both phenotypes prevalent in cells invading the surrounding tissue.Conclusion:Both neuroendocrine and epithelial phenotypes coexist in human SCLC tumours in vitro and in vivo and this persists in invading tumour cells. In patients, POMC secretion predicts poor survival and liver metastases, suggesting a crucial role of the neuroendocrine phenotype.

Targeting hypoxia in the treatment of small cell lung cancer

Small cell lung cancer (SCLC) is an extremely aggressive disease for which minimal therapeutic improvements have been made over the last few decades. Patients still rely on non-targeted, chemotherapeutic drugs complemented by irradiation. Although initial response is very good, the majority of SCLC patients invariably relapse with therapy-resistant tumours. Despite the link between pathologically low oxygen levels and therapy resistant tumours, hypoxia has gained little attention in the development of novel therapies for SCLC. In contrast, the advantages of targeting hypoxic cells in many other cancer types have been studied extensively. This review describes the reasons for targeting hypoxia in SCLC and outlines strategies undertaken to enhance hypoxic tumour cell death, including the use of bioreductive prodrugs, the targeting of HIF-1α and the induction of cell death through acidosis. Therapy directed towards hypoxic tumour regions has the potential to greatly enhance the response of SCLC tumours to current treatment regimens and represents an area of research in need of greater attention. Such research could lead to the much sought after development of targeted drugs against SCLC tumours.

Irradiation Decreases the Neuroendocrine Biomarker Pro-Opiomelanocortin in Small Cell Lung Cancer Cells In Vitro and In Vivo

Background Small cell lung cancer (SCLC) is an extremely aggressive disease, commonly displaying therapy-resistant relapse. We have previously identified neuroendocrine and epithelial phenotypes in SCLC tumours and the neuroendocrine marker, pro-opiomelanocortin (POMC), correlated with worse overall survival in patients. However, the effect of treatment on these phenotypes is not understood. The current study aimed to determine the effect of repeated irradiation treatment on SCLC cell phenotype, focussing on the neuroendocrine marker, POMC. Results Human SCLC cells (DMS 79) were established as subcutaneous xenograft tumours in CBA nude mice and then exposed to repeated 2Gy irradiation. In untreated animals, POMC in the blood closely mirrored tumour growth; an ideal characteristic for a circulating biomarker. Following repeated localised irradiation in vivo, circulating POMC decreased (p< 0.01), in parallel with a decrease in tumour size, but remained low even when the tumours re-established. The excised tumours displayed reduced and distinctly heterogeneous expression of POMC compared to untreated tumours. There was no difference in the epithelial marker, cytokeratin. However, there were significantly more N-cadherin positive cells in the irradiated tumours. To investigate the tumour response to irradiation, DMS79 cells were repeatedly irradiated in vitro and the surviving cells selected. POMC expression was reduced, while mesenchymal markers N-cadherin, β1-integrin, fibroblast-specific protein 1, β-catenin and Zeb1 expression were amplified in the more irradiation-primed cells. There were no consistent changes in epithelial marker expression. Cell morphology changed dramatically with repeatedly irradiated cells displaying a more elongated shape, suggesting a switch to a more mesenchymal phenotype. Conclusions In summary, POMC biomarker expression and secretion were reduced in SCLC tumours which regrew after irradiation and in repeatedly irradiation (irradiation-primed) cells. Therefore, POMC was no longer predictive of tumour burden. This highlights the importance of fully evaluating biomarkers during and after therapy to assess clinical utility. Furthermore, the gain in mesenchymal characteristics in irradiated cells could be indicative of a more invasive phenotype.

Glucocorticoid receptor-mediated apoptosis in small-cell lung cancer requires interaction with BCL2

Small-cell lung cancer (SCLC) tumours are highly aggressive. At the time of diagnosis, patients have often developed metastases, and overall prognosis is particularly poor, making effective treatment difficult. Novel mechanisms need to be identified as treatment targets. We have previously found low levels of the glucocorticoid receptor (GR) in SCLC cell lines and demonstrated that over-expression of GR increases tumour cell death both in vitro and in vivo. We hypothesise that low levels of GR impair its inhibitory effect on BCL2 and thus provide a survival advantage to SCLC cell lines. The mechanism behind GR-induced apoptosis is currently unknown; therefore, pro- and anti-apoptotic genes were investigated for their role in GR-mediated apoptosis signalling. We found that over-expression of wtGR via retroviral transduction causes the DMS 79 SCLC cell line to undergo caspase-mediated apoptosis within 72  h. Neither BAD nor BCL2L11 (BIM) mRNA and protein levels were affected by GR restoration implying that GR does not trigger apoptosis in the SCLC cell lines by up-regulating these pro-apoptotic genes. The anti-apoptotic BCL2 gene was significantly overexpressed in six SCLC cell lines and the BCL2 inhibitor ABT-737 increased apoptosis in all three cell lines tested. GR interacted with BCL2 in DMS 153, DMS 79 and COR-L42 cell lines, suggesting that a protein interaction between GR and BCL2 could play a role in GR-induced apoptosis. A deeper understanding of the molecular mechanism for increasing GR expression in SCLC could provide novel treatment strategies in the future.

Novel carbonic anhydrase IX-targeted therapy enhances the anti-tumour effects of cisplatin in small cell lung cancer

Small cell lung cancer (SCLC) has an extremely poor prognosis and methods of improving chemotherapeutic intervention are much sought after. A promising approach lies in inhibiting the tumour‐associated enzyme, carbonic anhydrase IX (CA IX), which supports tumour cell survival. The aim of this study was to assess the potential of CA IX inhibition using 4‐(3′‐(3″,5″‐dimethylphenyl)ureido)phenyl sulfamate (S4), for the treatment of human SCLC alone and in combination with cisplatin chemotherapy. Treating SCLC cell lines (DMS 79 and COR‐L24) with 100 µM S4 reduced viability in vitro and enhanced cell death when combined with 7 µM cisplatin, most prominently under hypoxic conditions (0.1% O2). When either cell line was grown as a xenograft tumour in nude mice, intraperitoneal injection of 50 mg/kg S4 alone and in combination with 3 mg/kg cisplatin led to significantly reduced tumour growth. Combination therapy was superior to single agents and response was greatly accentuated when administering repeated doses of cisplatin in DMS 79 tumours. The mechanism of therapeutic response was investigated in vitro, where S4 treatment increased apoptosis under hypoxic conditions in both DMS 79 and COR‐L24 cells. DMS 79 tumours receiving S4 in vivo also displayed increased apoptosis and necrosis. Combining S4 with cisplatin reduced both the area of hypoxia and CA IX‐positive cells within tumours and increased necrosis, suggesting hypoxia‐specific targeting. This study presents a novel, targeted approach to improving current SCLC therapy via inhibition of CA IX, which enhances apoptosis and significantly inhibits xenograft tumour growth when administered alone and in combination with cisplatin chemotherapy.

Cushing syndrome in a child due to pro-opiomelanocortin (POMC) secretion from a yolk sac tumor

CONTEXT Pituitary microadenomas and adrenal tumours are the most common causes for endogenous Cushing syndrome (CS) in children. CASE DESCRIPTION We describe a two-year old girl with Cushing syndrome due to ectopic pro-opiomelanocortin (POMC) production from an abdominal yolk sac tumor. Cortisol concentrations were elevated but adrenocorticotropic hormone (ACTH) concentrations were equivocal. The use of antibodies specifically detecting ACTH precursors revealed that plasma ACTH precursors were elevated. Additionally, an ACTH assay with a low cross-reactivity for precursors showed low concentrations of ACTH. Immunohistochemistry suggested POMC but not ACTH production by the tumour. CONCLUSION We describe a yolk sac tumour as a novel source of ectopic POMC production leading to CS in a young girl.

International survey on high- and low-dose synacthen test and assessment of accuracy in preparing low-dose synacthen

The short synacthen test (SST) is widely used to assess patients for adrenal insufficiency, but the frequency and protocols used across different centres for the low‐dose test (LDT) are unknown. This study aimed to survey centres and test the accuracy of ten different synacthen preparation strategies used for the LDT.

Abstract 4980: Inhibiting carbonic anhydrase IX targets hypoxic small cell lung cancer cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Small-cell lung cancer (SCLC) is an extremely aggressive disease, exhibiting acquired resistance to therapy and poor patient prognosis. Therapy resistance can in part be attributed to tumor hypoxia. Hypoxic SCLC cells up-regulate carbonic anhydrase IX (CA IX) in order to survive. CA IX is involved in pH maintenance of cells and presents a novel therapeutic target for SCLC. This study aimed to investigate the potential of inhibiting CA IX in SCLC as a novel therapeutic target. Hypoxic conditions (0.1% O2) increased CA IX expression over 24 hours in both COR-L24 and DMS 79 SCLC cells in culture. S4 belongs to a sulfamate class of carbonic anhydrase inhibitors and has shown anti-metastatic properties in breast cancer xenografts. Treatment with 30μM and 100μM S4 significantly decreased CA IX protein levels in hypoxic COR-L24 and DMS 79 SCLC cells, respectively (p<0.05 for both treatments). Exposure to S4 for 24 hours resulted in predominantly hypoxic cell death in both cell lines (p<0.05). Combining S4 with etoposide (0.3µM) and cisplatin (0.5µM) chemotherapeutic agents resulted in additional cell death, which was also seen under normoxic conditions (20% O2). Sub-cutaneous xenograft tumors were grown in nude mice using either COR-L24 or DMS 79 SCLC cells. Resultant tumors (1000mm3) expressed CA IX largely adjacent to regions of necrosis. Treating mice with 50mg/kg S4 on two consecutive days each week resulted in greater necrosis than saline controls, coupled with up-regulation of CA IX. Analyzing tumors for characteristic neuroendocrine (pro-opiomelanocortin) and epithelial (cytokeratin) markers revealed heterogeneous expression; however, this did not correlate with CA IX expression and is not altered in response to treatment. In summary, CA IX is upregulated in hypoxic SCLC cells and tumors. Inhibition of CA IX resulted in hypoxic cell death and had an additive effect to the response observed with chemotherapy treatment alone. This suggests that directly targeting hypoxic SCLC cells via CA IX inhibition holds promise in improving treatment outcome for this aggressive disease. Citation Format: Jennifer L. Bryant, Suzanne L. Meredith, Roben G. Gieling, Brian A. Telfer, Muhammad Babur, Claudiu T. Supuran, Kaye J. Williams, Anne White. Inhibiting carbonic anhydrase IX targets hypoxic small cell lung cancer cells. [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 4980. doi:10.1158/1538-7445.AM2014-4980

Abstract 1132: Irradiation promotes a mesenchymal switch in small cell lung cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Small cell lung cancer (SCLC) is the most aggressive form of lung cancer, with little improvement in therapy options and prognosis over the last few decades. Tumors initially respond well to chemotherapy and radiotherapy; however, when patients relapse, tumors have typically developed resistance to treatment. The aims of this study were to determine how irradiation treatment affects the phenotype of SCLC cells and in particular the neuroendocrine features of SCLC. Xenografts were established by subcutaneous injection of DMS 79 cells into nude mice, before exposure to ionising radiation (IR). Xenograft tumors and IR treated DMS 79 cells were analysed by immunohistochemistry and qPCR for markers of neuroendocrine, mesenchymal and epithelial phenotype. Circulating pro-opiomelanocortin (POMC), a neuroendocrine biomarker, was quantified by ELISA developed in our lab. Irradiation of xenograft tumors led to increased levels of the mesenchymal marker, N-cadherin, unchanged epithelial markers (cytokeratin and E-cadherin), as well as decreased and distinctly heterogeneous expression of POMC. We have previously described POMC as a novel biomarker in SCLC patients and xenografts. However, circulating POMC and tumor POMC protein levels and mRNA were drastically reduced in the irradiated animals. To determine whether the changes in phenotype were associated with resistance, DMS 79 cells were made irradiation resistant (IR-res) through repeated exposure to IR in vitro (total 21Gy). The IR-res cells acquired dramatic changes in morphology and adherence, showed increased proliferation and increased resistance to challenges with both small and larger doses of IR. In addition, cells exhibited increased expression of the mesenchymal markers; N-cadherin and β1 integrin. IR-res cells also showed a significant decrease in expression of the neuroendocrine marker POMC. However, there was no change in expression of other neuroendocrine markers; neuron-specific enolase (NSE), chromogranin A and neural cell adhesion molecule (N-CAM), or the epithelial marker E-cadherin. In summary, irradiation causes a distinct mesenchymal switch in DMS 79 small cell lung cancer cells in vitro and in vivo that does not resemble a classical epithelial to mesenchymal transition (EMT). Therefore, although irradiation is an important treatment paradigm in patients, our data suggests it may promote a phenotype that encourages metastasis. In addition, we have highlighted the need for caution when using biomarkers, as irradiation has modified POMC biomarker expression and secretion so that it no longer predicts tumor burden in relapsed SCLC. Citation Format: Suzanne L. Meredith, Jennifer L. Bryant, Muhammad Babur, Philip Riddell, Kaye J. Williams, Anne White. Irradiation promotes a mesenchymal switch in small cell lung cancer. [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 1132. doi:10.1158/1538-7445.AM2014-1132