- Open Access
Focal overexpression of CEACAM6 contributes to enhanced tumourigenesis in head and neck cancer via suppression of apoptosis
© Cameron et al.; licensee BioMed Central Ltd. 2012
- Received: 21 May 2012
- Accepted: 18 September 2012
- Published: 28 September 2012
Overexpression of CEACAM6 has been reported for a number of malignancies. However, the mechanism of how CEACAM6 contributes to cancer formation and its role in head and neck squamous cell carcinoma (HNSCC) remains unclear. Therefore, we examined the role of CEACAM6 in head and neck squamous cell carcinoma (HNSCC).
CEACAM6 expression was examined in normal squamous epithelia as well as a number of patient HNSCC samples and tumours derived from HNSCC cell lines injected into NOD/SCID mice. CEACAM6 expression was manipulated in HNSCC cell lines by shRNA-mediated CEACAM6 knockdown or virally-delivered overexpression of CEACAM6. The role of CEACAM6 in tumour growth and chemotherapeutic sensitivity was then assessed in vivo and in vitro respectively.
CEACAM6 expression was significantly increased in highly tumourigenic HNSCC cell lines when compared to poorly tumourigenic HNSCC cell lines. Moreover, HNSCC patient tumours demonstrated focal expression of CEACAM6. Functional investigation of CEACAM6, involving over-expression and knock down studies, demonstrated that CEACAM6 over-expression could enhance tumour initiating activity and tumour growth via activation of AKT and suppression of caspase-3 mediated cell death.
We report that CEACAM6 is focally overexpressed in a large fraction of human HNSCCs in situ. We also show that over-expression of CEACAM6 increases tumour growth and tumour initiating activity by suppressing PI3K/AKT-dependent apoptosis of HNSCC in a xenotransplant model of HNSCC. Finally, our studies indicate that foci of CEACAM6 expressing cells are selectively ablated by treatment of xenotransplant tumours with pharmacological inhibitors of PI3K/AKT in vivo.
- Tumour initiation
- Cleaved Caspase 3
CEACAM6 is a member of the cacinoembryonic antigen (CEA) family of immunoglobulin glycoprotein cell adhesion molecules (CAM) comprising at least 12 CEACAM members. CEACAMs are a diverse group of proteins which play major roles in cell-cell and cell-ECM adhesion and have been implicated in the control of cell proliferation, angiogenesis and tissue remodelling. More recently, CEACAMs have also been implicated in mediating tissue responses to pathogens. CEACAM6 is expressed at low levels in normal epithelial, endothelial and hematopoetic cells including granulocytes, T-cells and NK cells[2–4]. In contrast, CEACAMs are up-regulated in many epithelial malignancies including pancreatic, colorectal and breast cancers[5, 6]. The expression of CEACAM6 also correlates with the metastatic potential of some epithelial malignancies, suggesting that the altered expression of CEACAM6 may contribute to tumour progression. However, a definitive role for CEACAMs in tumourigenesis has not been formally proved. For example, CEACAM6 appears to affect the release of cytochrome-c from the mitochondria in response to cell detachment leading to the inhibition of caspase activation and hence, suppression of caspase induced apoptosis or anoikis in pancreatic cancer cells[8, 9]. These apoptotic-suppressive effects have been shown to be AKT-dependent in pancreatic cancer cells. Moreover, transgenic mice which overexpress members of the CEA family display colonic dysplasia. In contrast, CEACAM6 up-regulation is associated with an increase in apoptosis in acute lymphoblastic leukaemia (ALL), indicating that the apoptosis-modulating effects of CEACAM6 may be tumour-type-specific.
A recent transcriptomic profiling study comparing highly tumourigenic clonal variants of an established head and neck cancer squamous cell carcinoma (HNSCC) cell line with poorly tumourigenic clonal variants, identified a strong association between CEACAM6 expression and tumourigenic potential. Since an association between HNSCC and CEACAM6 expression has not been previously reported we now examine whether the over-expression of CEACAM6 is also present in human HNSCC samples.
Cell culture and patient tumours
All HNSCC cell lines were obtained from the ATCC and cultured as per ATCC recommendations (Sydney, NSW, Australia). Patient tumour samples were all confirmed as invasive squamous cell carcinoma (SCC) by a staff Pathologist (Princess Alexandra Hospital). Overall we examined 4 tongue SCC, 3 lip SCC and normal mucosae from all these patients. Normal human epidermal keratinocytes (HEKs) were isolated and cultured from neonatal foreskin samples following circumcision as described[12, 13]. Patient consent and approval by the Princess Alexandra Hospital Human Ethics Committee was obtained for all samples collected.
Reverse transcriptase and real-time PCR (rt PCR)
Total RNA was isolated from cell lines with the addition of trizol (Invitrogen, Melbourne, VIC, Australia) as per manufacturer’s instructions. Quantification and reverse transcriptase reaction was performed as previously described. The rtPCR CEACAM6 forward primer 5’ GACAGTTCCATGTATACCCG 3’ and the reverse primer 5’ACAGCATCCTTGTCCTCC 3’, were obtained from Sigma-Aldrich (Sigma-Aldrich, Sydney, NSW, Australia). The rtPCR reaction solutions were prepared and performed as per manufacturer’s instructions (Promega, Sydney, NSW, Australia). RtPCR reactions were performed as previously described.
Western blot analysis
Total cellular protein was isolated using RIPA buffer and quantified as previously described. Up to 20μg of protein was loaded onto a 10% SDS-PAGE, transferred onto PVDF membrane and probed as previously described. A 1/1000 dilution of anti-CEACAM6 antibody (Abcam, Sapphire Bioscience, Sydney, NSW, Australia), 1/1000 dilution of of anti-AKT or anti-phospho S473AKT and a 1/1500 dilution of the secondary anti-mouse Horse Radish Peroxidase (HRP) (GE Healthcare, Sydney, NSW, Australia) antibody was used to detect protein using chemiluminescence as per manufacturer’s instructions (Pierce, Rockford, IL, USA). Western blots were stripped as per manufacturers instruction (Thermo Scientific, Rockford, Il, USA) to re-probe with a 1/1000 dilution of β actin antibody (Sigma-Aldrich) and a 1:2000 dilution of the anti-Rabbit HRP (GE Healthcare) secondary antibody.
Cell proliferation and death assays in vitro
Bromo-deoxy uridine (BrdU) incorporation was used to estimate proliferation in vitro. For BrdU analysis, cells were plated at 104 cells per well in a 96 well plate (Sigma-Aldrich) 24 hours prior to incubation with BrdU. BrdU incubation and detection was performed as per manufacturer’s instructions (Roche, Sydney, NSW, Australia). In experiments examining the cytotoxic effects of the PI3K/AKT inhibitor, BGT226, cells were treated for 48 hours with varying doses of BGT226 following which viability was determined using the Celltiter assay kit (Promega Madison, WI, USA, G3580) as described. To measure basal levels of apoptosis in vitro Annexin V was added to a single cell suspension of Detroit 562 cells. The single cell suspension was isolated from the Detroit 562 cell line as previously described. The cells were stained with Annexin V Cy 5.5 as per manufactures instructions (BD Bioscience, Sydney, NSW, Australia) and analysed using FACSCanto Diva version 2.2 Software (BD Pharminogen, Sydney, NSW, Australia).
Generation of a stable knock down of CEACAM6 in the Detroit 562 cell line
For the generation of knock downs of CEACAM6, 2 microRNA interference (miR RNAi) sequences for CEACAM6 were made. The primers for the first miR RNAi sequence named miR CEA was, 5’ CACTGCCAAGCTCACTATTGAC 3’ for the top strand and bottom strand was 5’ GTCAATAGTGAGTGGCAGTG 3’. The other miR RNAi sequence for CEACAM6 was named miR CEA Dux, with a top strand of 5’ CCGGACAGTTCCATGTATACC 3’ and bottom stand of 5’ GGTATACATGGCTGTCCGG 3’ based on the shRNA sequence described in Duxbury et al.. The pLENTI 6.1 miR RNAi sequences for miR CEA, miR CEA Dux and control (lac Z) were generated and transduced into to the Detroit 562 cell line as per manufacturer’s instructions (GATEWAY pLENTI cloning system, Invitrogen).
Generation of a stable over-expression of CEACAM6 in the Detroit 562 cell line
The forward primer of 5 GGGGACAAGTTTGTACAAAAAAGCAGGCTCACCATGG GAGACCATGGGACCCCCCTCA3’ (attB1 site underlined) and reverse primer of 5’ GGGGACCACTTTGTACAAGAAAGCTGGGT GGGCTGCTATATCAGAGCCAC 3’ (attB2 site underlined) were used to generate full length CEACAM6 sequence from human epidermal keratinocytes (HEK) cDNA. The PCR conditions were as per manufactures instructions for Hifi taq (Promega). The CEACAM6 sequence was cloned into pDONR 221 (Invitrogen) using a BP reaction, then an LR reaction into pLV101G as per manufactures instructions (Invitrogen). The pLV101-Ceacam6 and pLV101 (control vector) Detroit 562 cell were generated as previously described.
Tumour initiation and tumour collection
Immunohistochemistry performed as previously described using CEACAM6 (Biogenex, Australia), PCNA (3.2 μg/ml, Sigma-Aldrich) and Cleaved caspase 3 (0.8 μg/ml, Promega) antibodies. Control antibodies were Rabbit IgG (DAKO, Copenhagen, Denmark) and Mouse IgG (DAKO). The percentage of positive cells (PCNA and Cleaved Caspase 3) was quantified as the number of positive cells per 40x magnified field of view from a minimum of 5 to 10 randomly selected fields using NIS-Elements BR3.1 image software (Nikon, Coherent Scientific, Adelaide, SA, Australia).
Student’s t test was used to assess the significance of differences between means of the different sample conditions.
CEACAM6 expression in HNSCC
The role of CEACAM6 in HNSCC tumourigenesity
In this study we report, for the first time, on the role of CEACAM6 in HNSCC. Previous work with keratinocytes and keratinocyte-derived SCC cells has shown that CEACAM6 is selectively expressed in differentiated keratinocytes and is highly expressed in a tumourigenic clonal variant of the Detroit 562 HNSCC cell line. In addition, other workers have reported that i) CEACAM6 overexpression occurs in variety of epithelial malignancies[5–7], ii) that CEACAM6 overexpression is associated with increased metastases, proliferation and the suppression of annoikis[7–9], iii) that CEACAM6 overexpression induces a src-dependent increase in AKT activity that suppresses gemcitabine sensitivity in pancreatic cancer cells and finally, iv) a transgenic model of CEA-overexpression suggests CEACAM6 overexpression can contribute to the development of colonic dysplasia. We now extend these findings and report that CEACAM6 is focally overexpressed in a large fraction of human HNSCCs in situ. The heterogeneous pattern of CEACAM6 overexpression is also evident in established HNSCC cell lines in vitro and in vivo. Moreover, we show that over-expression of CEACAM6 increases tumour growth and tumour initiating activity by suppressing PI3K/AKT-dependent apoptosis of HNSCC in a xenotransplant model of HNSCC. Finally, we show that foci of CEACAM6 expressing cells are selectively ablated by treatment of xenotransplant tumours with pharmacological inhibitors of PI3K/AKT in vivo.
A novel finding in the present study is the observation that CEACAM6 is focally overexpressed in the majority of HNSCCs examined. Whilst the sample size examined was small it highlights an important issue that has important biological and clinical implications. Specifically, intratumoural heterogeneity is a major contributor to the emergence of drug resistance and tumour recurrence. Consistent with this, our data suggest that focal overexpression of CEACAM6 is indicative of sensitivity of human HNSCC to selective cytotoxic drugs. In this regard two observations relating to CEACAM6 are relevant. Firstly, knockdown or overexpression of CEACAM6 resulted in a decrease and increase in tumourigenic activity in SCC cells in vivo respectively. Secondly, CEACAM6 has been shown to modulate the cytotoxic effects of conventional chemotherapeutics such as gemcitabine in pancreatic cancer cell lines and in the present study we showed that CEACAM6 could mediate sensitivity to new targeted agents such as the PI3K inhibitor, BGT226. It is noteworthy that the modulation of gemcitabine sensitivity is also mediated via a src and PI3K/AKT-dependent pathway. These data indicate that whilst CEACAM6 may invoke pro-survival responses in cancer cells by activating the PI3K/AKT pathway this same pathway could be selectively targeted by specific cytotoxic drugs. Thus, the presence of CEACAM6+ve foci would be predicted to bestow selective sensitivity against certain chemotherapeutic treatments (eg: gemcitabine or PI3K inhibitors). Proof of principle for this hypothesis is shown by the reduction in phospho-S437 AKT induced by knockdown of CEACAM6 and the loss of CEACAM6+ve foci in tumours treated with cytotoxic doses of PI3K inhibitors. Thus, CEACAM6 could be used to predict PI3K inhibitor sensitivity. Moreover, the observation that CEACAM6 expression correlates with metastatic potential[8, 20–22] would suggest that, in chemotherapy-naive tumours, the presence of CEACAM6+ve foci could serve as a prognostic marker of poor outcome and in this instance targeting CEACAM6/PI3K/AKT pathways could be exploited therapeutically. Supporting this, is a recent study, by Blumenthal et al., demonstrating that the addition of antibodies that inhibited the binding of CEACAM6+ve breast cancer cells to endothelial cells reduced tumour cell invasion. Finally, intratumoural heterogeneity can arise through a number of mechanisms such as the evolution of variant cells from a common clonal precursor, micro-environmental influences, stochastic processes or tissue/cell plasticity. The present study suggests that the focal pattern of CEACAM6 expression, in tumours, is derived from a specific clonal progenitor within the tumour rather than being transiently induced by the local environment. This is based on the observation that CEACAM6+ve and –ve cells persist in long term tissue culture models, consistent with an heritable mechanism (eg: genetic or epigenetic).
Whilst CEACAM6 clearly has the capacity to contribute to drug resistance and tumour recurrence it is clear that other factors also contribute to drug resistance and tumour recurrence. This is supported by our observation that targeted inhibition of the CEACAM6/PI3K/AKT pathway in SCC cells induced killing of 50% of the total HNSCC cells. Similarly, we have identified clonal variants of HNSCC cells that express very low levels of CEACAM6 yet still retain tumourigenic potential. Moreover, we show that the knockdown of CEACAM6 results in a decrease, but not an ablation, of tumour initiating activity or tumour growth. Thus, CEACAM6 likely represents one factor, of many, that can modulate tumour growth and tumour initiating activity. This is entirely consistent with the emerging importance of intratumoural heterogeneity. We previously reported that HNSCC display intratumoural heterogeneity that was reflected in histomorphologically and transcriptomically distinct clonal variants[11, 14]. We showed that clonal variants of HNSCC cells could persist in vitro in established cell lines and displayed significant differences in tumour initiating activity and drug resistance[11, 13, 14]. Several groups have now definitively shown, by single cell sequencing, that tumours comprise multiple genetically distinct clonal populations[23–27]. Emerging, clinical and molecular data unequivocally show that the presence of intratumoural heterogeneity, exemplified by focal CEACAM6 overexpression in HNSCC cells, is a major contributor to tumour drug responses and patient outcomes.
Earlier work by Duxbury, suggests that the major contribution of CEACAM6 to tumour growth and tumour initiating activity is mediated via suppression of anoikis. Anoikis is a form of apoptosis induced by loss of cell-cell/EMC contact. Thus, anoikis may be more relevant to a 3 dimensional tumour environment rather than an in vitro cell monolayer system.. Supporting this, we found that the in vivo effects of CEACAM6 over-expression/knockdown were not reflected by the in vitro effects of CEACAM6. For instance, CEACAM6 over-expression/knockdown had modest and inconsistent effects on apoptotic rates in vitro. However, over-expression of CEACAM6 significantly reduced caspase-3 dependent apoptosis of HNSCC cells in a xenotransplant model. Anti-apoptotic activity is commonly viewed as tumour promoting and hence the anti-apoptotic activity of CEACAM6 would suggest it has tumour promoting (oncogenic) activity. CEACAM6-mediated inhibition of apoptosis in vivo therefore contributes in part, or wholly, to the ability of HNSCC cells to initiate a tumour in a xenotransplant model of HNSCC. In addition, CEACAM6 over-expression also contributes in part, or wholly, to the increased tumour growth in a xenotransplant model of HNSCC. Based on these findings, it is reasonable to speculate that focal patches of CEACAM6 expressing cells within HNSCC may reflect the presence of a subpopulation of cells with a greater potential for recurrence/metastasis than CEACAM6-ve subpopulations of HNSCC cells.
In conclusion, our study shows that CEACAM6 is focally overexpressed in a large fraction of human HNSCCs in situ and contributes to tumour growth and tumour initiating activity. The effect of CEACAM6 on tumour growth and initiation is mediated via suppression of PI3K/AKT-dependent apoptosis of HNSCC in a xenotransplant model of HNSCC. Finally, our studies show that CEACAM6+ve tumours, or tumour foci, are selectively sensitive to treatment with pharmacological inhibitors of PI3K/AKT in vivo.
This work was supported by a PhD scholarship awarded to SC by the Garnett Passe & Rodney Williams Memorial Foundation. NS is supported by a senior research fellowship awarded by the Cancer Council Queensland. This work was also supported by a research grant awarded to NS (#455929, #569689) from the Australian NHMRC: Cancer Council QLD, #631479 and a practitioner fellowship to AG from the Cancer Collaborative Group.
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