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Characterization of Intracellular and Extracellular Metabolites in Squamous-Cell Carcinoma (SCC) !
M. Bernardo*, S. Kamlarz**, J. Reyes**, Dr. A. Alt-Holland** and Dr. J. Baleja***!
School of Arts & Sciences*, School of Dental Medicine**, Sackler School of Biomedical Sciences***!
Mark Bernardo!Tufts University, A&S 2015!Baleja Lab – MV [email protected]!
Contact!1. Images produced by S. Kamlarz, J. Reyes, E. Bingham & Dr. A. Alt-‐Holland (School of Dental Medicine).
Used with permission. 2. hmp://www.canceralia.eu/wp-‐content/uploads/RhoGTPases.png 3. PDB ID: 2CKQ; image rendered using PyMOL 4. de Molina, Ana Ramirez, et al. "Overexpression of Choline Kinase Is a Frequent Feature in Human
Tumor-‐derived Cell Lines and in Lung, Prostate, and Colorectal Human Cancers." Biochemical and Biophysical Research Communica4ons 296.3 (2002): 580-‐83. Web.
5. Lamarre, Simon G., et al. "Formate: An EssenAal Metabolite, a Biomarker, or More?" Clinical Chemistry and Laboratory Medicine 51.3 (2013): n. pag. Web.
References!
Squamous cell carcinoma (SCC) is the cancer of the squamous cells found in the epithelium. Common prophylactic measures are proven to be ineffective in decreasing risk of SCC development. Nevertheless, it is known that loss of the cell-cell adhesion molecule and known tumor suppressor, E-cadherin (E-cad), is a hallmark of invasive SCC.!
Introduction!
Background!
- Two cell lines were used: E-cadherin competent SCC (II-4) and E-cadherin suppressed (II-4-Ecad-) cells. Cell lines derived from ras-transformed human keratinocytes (HaCaT). - Cells were grown in RPMI-1640 (HEPES modified) media for 48 hrs. 1.5 million II-4 and II-4-Ecad- cells were collected and plated in triplicate. - Intracellular metabolites were collected through methanol extraction. Conditioned media was also collected. - 600 MHz 1H-NMR spectroscopy and CHENOMX were used to determine the concentrations and identity of intracellular and extracellular metabolites. Concentrations were normalized by sum to account for variable extraction efficiency. MetaboAnalyst was used for statistical analysis.
Methods and Materials!
Conclusions!
Results!
Aim!To determine if there are metabolic differences between II-4-E-cad+ and II-4-Ecad- SCC, and characterize the intracellular and extracellular metabolites via 1H-NMR spectroscopy to establish a potential biomarker for invasive squamous cell carcinoma. !
Fig 1. E-cadherin-based adherens junction and E-cadherin loss in SCC1!
A.!
B.!
C. !
D.!
Normal Epithelium!
Cancerous Epithelium!
II-4 (E-cad+)!
II-4-Ecad-!
IF: DAPI/E-‐CAD
IF: DAPI/E-‐CAD
Cell-cell adhesion as facilitated by E-cadherin in normal epithelium (A). Cancer cells lose cell-cell adhesion due to loss of E-cadherin (B). Immunofluorescence (IF) staining of E-cadherin and cell nuclei. Defined E-cadherin expression in cell-cell junctions leads to compact cell cluster (C). Loss of E-cadherin leads to cell dispersion (D). !
Results!Fig 2. Stacked 1H-NMR spectra overlay of II-4 & II-4-E-cad- with intracellular
metabolites of interest!
1
2a
2b 3
4 5a 9
11
8
6
5b
7
14 13 12
5c
10
1H-NMR (600 MHz) spectra of II-4 (E-cad+) (top spectrum) and II-4-E-cad- (bottom spectrum). The spectra show different peak ratios which suggest varying metabolite concentrations. 1 branched amino acids, 2a&2b lactate, 3 alanine, 4 acetate, 5a glutamine/glutamate, 5b glutamate, 5c glutamine, 6 dimethylamine, 7 creatine/creatine phosphate, 8 glycerophosphocholine/phosphocholine, 9 myo-inositol, 10 glycine, 11 glutamine/glutamate/glutathione, 12 glucose, 13 NAD+, 14 aromatic amino acids.!
Fig 3. Phosphocholine (PCho) and other statistically significant II-4 & !II-4-E-cad- intracellular metabolites!
A.! B.!
Phosphocholine
Metabolite statistical significance determined via t-test and displayed as heat maps for both II-4 and II-4-E-cad-; red shows high relative intracellular concentrations, while blue shows low relative cellular concentrations (A). The red spectrum denotes II-4-E-cad- and the blue spectrum denotes II-4, and (B) shows an increase in II-4-E-cad- phosphocholine (PCho) levels (p=0.001, FC=2). !
* denotes p<0.05 !** denotes p<0.005
Fig 4. The Kennedy pathway and choline kinase!
Phosphocholine is involved in the Kennedy Pathway (lipid membrane synthesis) and accumulation of PCho in II-4-E-cad- may suggest: 1) increased choline uptake, 2) upregulation of choline kinase (ChoK), 3) downregulation of choline phosphate cytidyl-transferase (A).2 Homodimeric structure of choline kinase with PCho (orange) in binding sites (B).3 Upregulation of choline kinase has been implicated in a variety of malignant cancers.4!
1.!
2.!
3.!
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Glucose Lactate
Normalized
Con
centraGo
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Clear Media
Ecad+ (avg)
Ecad-‐ (avg)
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Phe Val Thr Trp Met Leu Ile Lys His
Normalized
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centraGo
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E-‐cad+ (avg)
E-‐cad-‐ (avg)
A.! B.!
Fig 5. Conditioned media analysis (extracellular metabolites)!A.! B.!
* denotes p<0.05 !** denotes p<0.005
C.!
0.000
0.010
0.020
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Choline
Normalized
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centraGo
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Ecad+ (avg)
Ecad-‐ (avg)
A.!
Media concentrations of glucose are similar in both II-4 and II-4-E-cad-, which suggest same glucose uptake; lactate production is also similar (A). Media concentrations of essential amino acids also show similar uptake for both cell types with exception of threonine (B). Choline media concentrations are relatively similar for both cell types, thus phosphocholine accumulation may not be a result of increased choline uptake (C). !
Fig 6. Statistically significant extracellular metabolites & potential link to one-carbon metabolism!
Statistically significant metabolites displayed on heat maps: formate, glycine, threonine and valine levels increase in II-4-E-cad- media, which suggests II-4-E-cad- increased production of these metabolites (A). Accumulation of formate in II-4-E-cad- could suggest altered one-carbon metabolism, since formate is a key intermediary metabolite. Methyl group transfers are vital to nucleotide and amino acid synthesis, among other pathways. Glycine is also implicated in these pathways as a byproduct of serine metabolism (B).5!
- There are distinct metabolic differences between SCC cells that have lost cell-cell adhesion (II-4-E-cad-) and SCC cells with cell-cell adhesion (II-4-E-cad+). - A possibly affected pathway is phospholipid synthesis and breakdown (Kennedy Pathway) due to accumulation of phosphocholine in II-4-E-cad-. - One-carbon transfers (methyl group) may also be affected due to increased production of glycine and formate. !
Current & Future Directions!- Establish a direct link between phosphocholine accumulation and loss of cell-cell adhesion.!- Assess enzyme expression & activity - enzymes involved in choline metabolism and methyl group transfers.!- Chloroform/MeOH extraction to assess lipid concentrations of II-4-E-cad- cells.!- CURRENTLY: observed Dab2 downregulation in tandem with E-cadherin loss. Assessing variable metabolic profiles of SCC based on degree of Dab2 loss.!
B.! One-carbon pathway centered on formate!
Acknowledgements!- Mentor: Dr. J. Baleja!- Alt-Holland Lab: Dr. A. Alt-Holland, S. Kamlarz, J. Reyes!- Anne Moore & the Summer Scholars Program!- Nathan Gantcher Student Summer Scholar Program!
