To identify new potential cancer diagnostic or therapeutic targets, suppression subtractive hybridization (SSH) transcript profiling was performed between normal human cultured bronchial epithelial cells (HBE) and the metastatic small cell lung carcinoma cell line H526 (Difilippantonio et al., 2003).

The quality of the SSH library was confirmed by Northern blot analysis, indicating that 69% (236/342) of the clones were differentially expressed in the tumour cell line. In an initial evaluation we used a limited panel of cell lines including HBE cells, as well as lung tumour cell lines 2170, H526, and D51, representing the three major subtypes (squamous cell carcinoma, adenocarcinoma, and small cell lung carcinoma). Thirty-one cDNAs scored as differentially expressed in at least two of the tumour cell lines were analysed more extensively on an extended panel of cell lines including small airway epithelial cells (SAE), four immortalized HBE cells, and 32 lung cancer cell lines. The DNA sequences of isolated cDNA fragments were then determined and compared with those deposited in the GenBank database.

Based on our interest in further examining those transcripts with no homology to any known gene, 6 partial cDNAs representing transcripts that were abundantly expressed in normal lung epithelial cells but were absent or under-represented in the majority of lung tumour cell lines were selected for further analysis. These cDNA fragments were sent to the German Resource Center of the Human Genome Project (RZPD, Heidelberg, Germany) for screening of human cDNA libraries enriched in full-length cDNAs. Three of those cDNA fragments represented the same unknown gene. Screening of the human lung, trachea, and primary keratinocyte cDNA libraries yielded 13 positive clones out of which 9 were confirmed positive by Southern blot analysis. Out of 5 clones containing the longest inserts determined by NotI/SalI-restriction digest, clones DKFZp404H1043, DKFZp404B1018, and DKFZp719J2437 were identified as candidate full-length cDNA clones. Clone DKFZp404H1043 (designated H1043) with cDNA insert of 1057 bp was chosen for further characterization. The insert size was in accordance with the transcript size estimated by Northern blot analysis.

On the nucleotide level no homology to any known gene was found. Analysis of the predicted protein sequence revealed an S100-specific EF-hand Ca²⁺-binding domain as well as a high homology to the S100A13 calcium-binding protein. Therefore, we designated the transcript S100A14 and submitted it to the NCBI GenBank database under Accession No NM_020672 (LocusID LOC57402).

To verify the full-length cDNA sequence of S100A14 we performed 5’ RACE on human colon cDNAs since this organ showed the most abundant expression of the transcript in our Northern blot analysis. It provided additional 5’ UTR sequence, not present in the H1043 clone. The predominant cDNA product was 1067 bp in length.

The cDNA was predicted to contain an open reading frame (ORF) of 104 amino acids (nt 109-423), a 5’ untranslated region (UTR) of 108 bp, and a 3’ UTR of 644 bp. The putative ATG initiation codon was embedded in a strong Kozak consensus sequence (CACCATGG) (Kozak, 1996) and was preceded by an in-frame stop codon. A polyadenylation signal (nt 1028-1033) and a poly(A)-tail beginning after nucleotide 1053 oriented the fragment and provided further evidence for the predicted ORF. The nucleotide sequence and the predicted open reading frame of S100A14 are shown in Fig. 3.

Computer-assisted analysis of the deduced amino acid sequence revealed an S100-specific EF-hand Ca²⁺-binding domain at the N-terminus (aa 33-45) consisting of 13 amino acids. The second putative canonical EF-hand motif has been deduced from the secondary structure and hydrophobicity analysis of the protein. It contained only 2 out of 6 conserved residues (Asp76 and Glu80) and might start at Gly72 ending at Ser83. In addition, the protein was predicted to contain a N-glycosylation site (aa 75-78), a protein kinase C phosphorylation site (aa 94-96), 5 casein kinase II phosphorylation sites (aa 16-19, 42-45, 73-76, 7780, 83-86), and a N-myristoylation site (aa 2-7).

The calculated molecular weight of S100A14 is 11 662 Da and the isoelectric point is 4.99. Protein secondary structure analysis indicated two helix-loop-helix structural motifs characteristic of calcium-binding sites. There was an extended hydrophilic loop at the extreme N-terminus in contrast to other S100 family members.

Fig.   3 Nucleotide and deduced amino acid sequence of the human S100A14 gene. The predicted start codon and polyadenylation signal are underlined. An asterisk indicates the putative stop codon. Numbers to the left correspond to the single-letter amino acid sequence. Numbers to the right correspond to the nucleotide sequence.

The deduced protein of S100A14 shares 68% similarity and 38% identity to S100A13 (Accession No Q99584) being the closest human match. Significant similarity was also found with S100A4 (62% similarity; 30% identity), S100A2 (60% similarity; 30% identity), S100A10 (58% similarity; 31% identity), and S100A9 (55% similarity; 34% identity), as illustrated in Fig. 4 . Furthermore, in the SwissProt database we identified an apparent mouse orthologue, 1110013O05RIK protein (Accession No Q9D2Q8), showing 97% similarity (93% identity) using the human S100A14 amino acid sequence as a query. A comparison between the S100A14 cDNA and RIKEN 1110013O05 (1110013O05Rik) cDNA (GenBank Accession No NM_025393) revealed 87% identity.

Fig.   4 Alignment of the predicted amino acid sequences of the human S100A14 and the mouse orthologue 1110013O05RIK with the most homologous S100 family members. Homology is represented by shading of the amino acid identities. Identical amino acid residues are indicated as black boxes whereas similar amino acids are indicated as gray boxes.

To characterize S100A14 expression in tumour cell lines from other tissues we extended the expression analysis to normal human cultured mammary epithelial cells, four immortalized epithelial cell lines, and 31 different human tumour cell lines as well as ten mouse and rat cell lines. The complete list of the examined cell lines is given in Table 2 and Table 3.

Cell lines

Histopathology

S100A14

HBE

Normal human bronchial epithelial cells

+++

SAE

Normal human small-airway epithelial cells

+++

H378

SCLC

–

H82

SCLC

–

COLO 677

SCLC

RT-PCR

H446

SCLC

–

CPC-N

SCLC

–

DMS-79

SCLC

+

H209

SCLC

RT-PCR

DMS-114

SCLC

–

H187

SCLC

RT-PCR

N417

SCLC

–

H526

SCLC metastatic

–

COLO 668

Brain metastasis of SCLC

–

SHP77

Large cell variant of SCLC

–

COLO 699

ADC

–

DV-90

ADC

+

H322

ADC

+

D51

ADC

RT-PCR

D54

ADC

RT-PCR

D117

ADC

–

H125

ADC

–

H23

ADC

–

A549

ADC

RT-PCR

H2228

ADC

RT-PCR

H2030

ADC

–

H157

SCC

–

H2170

SCC

+

H226

SCC mesothelioma

–

H596

Lung Ca

–

D40/97

Lung Ca

+

A427

Lung Ca

–

BEN

Lung Ca, lymph node metastasis

–

D97

LCLC

–

HBE-E6/E7

HBE immortalized with HPV-16

++

9442 (BET-1A)

HBE immortalized with SV40 early region

++

BEAS-2B

HBE immortalized with SV40 early region

++

YP44

HBE immortalized with SV40 early region

++

DU 145

Prostate Ca

–

PC-3

Prostate Ca

–

HMEC

Normal human mammary epithelial cells

++

HMEB

HMEC immortalized with telomerase and SV40

+

MCF-10A

Spontaneously immortalized mammary epithelial cells

++

MCF-7

Breast Ca

–

SK-BR-3

Breast Ca

–

MDA-MD-231

Breast Ca

–

Kato III

Metastasis of gastric Ca

++

HT-29

Colon Ca

+

SW-480

Colon Ca

–

HCT-116

Colon Ca

+

CaCo-2

Colon Ca

++

WiDr

Colon Ca

+

Lovo

Colon Ca

+++

CX-2

Colon Ca

+

HRT-18

Colon Ca

++

EJ

Bladder Ca

–

HeLa

Cervical Ca

–

Daudi

Burkitt’s lymphoma

–

K562

Chronic myelogenous leukemia

–

HL-60

Promyeolocytic leukemia

–

MOLT-4

Lymphoblastic leukemia

–

Raji

Burkitt’s lymphoma

–

G361

Melanoma

–

SKOV-3

Ovarian Ca

–

OVCAR-3

Ovarian Ca

+

HT 1080

Fibrosarcoma

–

HEP-2

Head and neck Ca

–

D36-1/95

Head and neck Ca

+

D36-2/95

Head and neck Ca

+

D6/95

Head and neck Ca

+

D3/02

Head and neck Ca

–

IMR-90

Fetal lung fibroblasts

–

HAKAT

Immortalized keratinocytes

+

HEK 293

Human embryonic kidney epithelial cells

–

Table  2 Expression of S100A14 mRNA in human cancer cell lines. S100A14 transcript level was examined by Northern blot and RT-PCR analysis. The expression level was determined by semiquantitative scoring as follows: (+++) strong expression, (++) moderate expression, (+) weak expression, (RT-PCR) transcript not detectable by Northern blot but detected by RT-PCR, (–) negative expression. The tumour cell lines histopathology: SCLC, small cell lung cancer; ADC, adenocarcinoma; SCC, squamous cell carcinoma; LCLC, large cell lung cancer; Ca, carcinoma.

The S100A14 mRNA is most abundant in cells cultured from normal human epithelial cells, but is absent from the vast majority of cancer-derived cell lines. Interestingly, most of the immortalized human cell lines also show significant S100A14 mRNA expression. The S100A14 mRNA level is relatively high in most of the colon tumour cell lines. We did not detect S100a14 in any mouse or rat cell line examined using a mouse S100a14 cDNA sequence as a hybridization probe.

Cell lines

Histopathology

S100a14

COS-7

Monkey kidney fibroblasts

–

RAW 264.7

Mouse macrophagoid immortalized cells

–

L-cells

Mouse immortalized fibroblasts

–

L-Wnt 3 cells

L-cells transfected with Wnt-3A

–

C57MG

Mouse mammary epithelial cells

–

ROSE 199

Rat ovarian surface epithelial cells

–

ROSE A2/1

ROSE 199 cells transfected with KRas

–

ROSE A2/5

ROSE 199 cells transfected with KRas

–

208F

Immortalized rat fibroblasts

–

FE-8

HRas-transformed derivative of 208F

–

IR-4

208F cells transfected with IPTG-inducible Ras

–

Table  3 Expression of S100A14 mRNA in other mammalian cell lines

We examined the expression pattern and transcript size of S100A14 in normal human tissues by Northern blot hybridization using a cDNA probe corresponding to the complete protein coding region. The transcript of approximately 1.1 kb was expressed most abundantly in colon ( Fig. 5 A). Moderate levels of S100A14 mRNA were detected in thymus, kidney, liver, small intestine, and lung. A very low expression level was detected in heart where a slightly longer transcript of approximately 1.35 kb was observed. No expression could be detected in brain, skeletal muscle, spleen, placenta, and peripheral blood leukocytes. Additionally, S100A14 was expressed in normal breast, ovary, prostate, rectum, stomach, thyroid, and uterus as determined by hybridization to the Cancer Profiling Array ( Fig. 5 B).

To confirm the mRNA expression data, we analysed a panel of normal human tissues by immunohistochemistry using a polyclonal anti-S100A14 antibody raised in rabbit. The S100A14 protein expression was predominantly associated with various epithelia as well as gland cells ( Table 4 ). The expression pattern was virtually consistent with the S100A14 mRNA expression in normal human tissues. The exceptions were brain and placenta, showing a weak expression of S100A14 protein, in contrast to the absence of the transcript in Northern blot analysis.

Fig.   5 Expression of the S100A14 mRNA in normal human tissues A:A human 12-lane Multiple Tissue Northern Blot (BD Biosciences) containing 2 µg poly(A)⁺ RNA in each lane was hybridized with a ³²P-labelled S100A14 cDNA probe. -actin was assayedas a loading control. B: Expression of S100A14 mRNA in additional normal human tissues examined by hybridization to the Cancer Profiling Array (BD Biosciences).

These results suggest that there is a very low S100A14 expression in these tissues and a not sufficient sensitivity of Northern blot to detect low abundant transcripts.

Table  4 Expression of the S100A14 protein in normal human tissues

Since S100A14 mRNA was not expressed in most of the tumour-derived cell lines, we hypothesized that its expression might be suppressed also in primary tumour tissue. To examine its expression in primary tissue we applied a commercially available Cancer Profiling Array, which contains cDNAs from 241 tumours and corresponding normal tissues from individual patients. This analysis, however, revealed a varied expression of the gene in different tumours ( Fig. 6 A). Significant overexpression was detected in ovary (78.6%), breast (62%), and uterus (45.2%) tumour tissues. Also lung (38.1%), prostate (25%), and thyroid gland (16.7%) tumours showed up-regulated levels of S100A14 transcript. In contrast, marked underexpression was found in kidney (70%), rectum (50%), and colon (40%) tumours and to lesser extent in stomach tumours (18.5%). The expression status in cervix, small intestine and pancreas tumours could not be evaluated because of the limited number of samples available on the array. The schematic presentation of S100A14 expression in normal and tumour tissues is shown in Fig. 6 B.

Fig.   6 Expression profile of S100A14 in human tumours A: The Cancer Profiling Array containing SMART-amplified cDNA from tumour (T) and corresponding normal (N) tissues from individual patients was hybridized with ³²P-labelled S100A14-specific probe. Hybridization to ubiquitin cDNA (top and bottom) and E.coli DNA (middle) is shown on the right side of the array. Human cancer cell lines (no detectable signal) are spotted on the right. B:The schematic presentation of S100A14 expression in normal and tumour tissues determined by hybridization to the Cancer Profiling Array.Data are expressed as percentage of up-regulated, down-regulated and normal samples and normalized to all spots on the membrane to correct for RNA quantity and integrity. The grey bars represent normal expression level, the black and white bars denote up-regulated and down-regulated expression, respectively. Signal intensities were quantified after several exposure times to compensate for differences in mRNA expression between different organs.

Our transcript expression analysis in primary tumour tissues suggested that S100A14 is not a simple suppressor of tumour cell growth as might have been concluded from the cell line model system. It is possible that its altered expression is a result of, or otherwise associated with, tumour progression. To shed light on the association of S100A14 protein with malignant transformation and to verify the RNA expression data we used immunohistochemistry to investigate its expression in tumour tissues.

S100A14 protein was examined in 120 histological sections of human lung cancer and 10 specimens of normal human lung epithelium. The cohort of tumour samples included 62 cases of adenocarcinoma (51.7%) and 58 cases of squamous cell carcinoma (SCC) (48.3%). The small number of samples precluded a separate analysis of large cell lung cancer (LCLC) tumour specimens. We observed a low cytoplasmic S100A14 staining in normal lung epithelium (score 0 to 1+; Fig. 7 ). Therefore, we approved a scoring of 2+ and 3+ as indicative of protein overexpression for our analysis. Scores 0 or 1+ were considered as physiologically normal expression.

Fig.   7 S100A14 protein is overexpressed in primary lung tumour tissue in comparison to normal lung tissue. Immunohistochemical staining of S100A14 protein in tissue sections of normal lung pneumocytes (left, indicated by an arrow) and lung squamous cell carcinoma (right). Specimens were stained with an anti-S100A14 antibody. Photographs were taken with a 40x objective.

Positive staining was assessed not only as the degree of staining intensity but also as the proportion of cells with staining of both plasma membrane and cytoplasm as compared with those of cytoplasmic staining alone.

S100A14 immunoreactivity in tumour tissue scored positive in 68.3% (82/120) of cases thus validating its up-regulation in this tumour type ( Fig. 7 ). The S100A14 staining was restricted to the cancerous epithelial cells of the tissue. Next, S100A14 expression was analysed in relation to clinicopathological criteria. No significant correlation was found between S100A14 positivity and gender, age, histological subtype, tumour size, number of positive lymph nodes, and histological grade.

We found that 57.3% (47/82) of S100A14-overexpressing lung tumours displayed cytoplasmic and membranous expression relative to exclusively cytoplasmic S100A14 staining in normal lung epithelium (p = 0.006; Fig. 8).

Fig.   8 S100A14 localizes to plasma membrane in lung tumour tissue. Immunohistochemical analysis of S100A14 protein in sections of normal bronchial epithelial cells (left) and lung squamous cell carcinoma (right). Panels show high power microscopy (magnification, 40x), demonstrating cytoplasmic staining of S100A14 in normal bronchial epithelial cells (left, indicated by an arrow) and distinct plasma membrane-associated staining of S100A14 in lung carcinoma cells (right).

Moreover, we observed a significant association (p = 0.048) between higher-grade tumours (grade 3) and S100A14 subcellular distribution. 50% (27/54) of high-grade tumours displayed S100A14 protein localized both to the cytoplasm and the plasma membrane. Another relation was found between lung tumour histology and subcellular distribution of S100A14. Cytoplasm and plasma membrane-localized S100A14 staining was associated with 61.8% (34/55) of SCC (p = 0.02) compared to 38.2 % (20/58) of adenocarcinoma tumours, the latter detecting predominantly cytoplasmic S100A14 (65.5%; 38/58). S100A14-negative tumours were not included in the statistical analysis of the subcellular distribution of S100A14. These results are summarized in Table 5 .

Table  5 Association of S100A14 protein expression in lung tumours with clinicopathological factors

A significant up-regulation of S100A14 mRNA in breast tumours prompted us to investigate this tumour type by immunohistochemistry. We examined S100A14 protein expression in 93 breast tumour samples, 14 lymph node metastases and 10 normal mammary epithelia. In our cohort of breast tumours were 77 invasive ductal carcinomas (IDC), 14 invasive lobular carcinomas (ILC) and 2 cases of mixed type (IDC+ILC).

Normal mammary epithelium stained rather weakly for S100A14 protein with cytoplasm-localized staining (score 1+; Fig. 9 ). Therefore we applied the same scoring as for lung tumours, i.e. scores 0 to 1+ were considered as normal physiological expression, whereas scores of 2+ and 3+ were assessed as positive and indicative of protein overexpression. S100A14 positivity was detected in 68.8% (64/93) of breast tumour samples and 71.4% (10/14) of lymph node metastases ( Fig. 9 ). Clinical relevance of S100A14-overexpressing tumours was investigated only in IDC cases due to small sample number of other tumour subtypes. No significant correlation with gender, age, histological subtype, tumour size, number of positive lymph nodes or histological grade was observed.

Fig.   9 S100A14 protein is up-regulated in primary breast carcinoma relative to normal breast tissue. Immunohistochemical analysis of S100A14 protein in normal breast ductal epithelial cells (left) and ductal carcinoma epithelial cells (right). Specimens were stained with an anti-S100A14 antibody. Photographs were taken with a 20x objective.

Interestingly, we found a significant correlation between a high S100A14 level and ERBB2 overexpression (p = 0.04). 82.9% (29/35) of ERBB2-overexpressing tumours (score 2+ and 3+) had up-regulated S100A14 expression as well ( Fig. 10 ). To further substantiate this finding, we separately examined 31 ERBB2-overexpressing breast tumours of score 3+. We found that 90.3% (28/31) of ERBB2-positive tumours were also S100A14-positive.

Fig.   10 S100A14 overexpression correlates with ERBB2 overexpression in primary breast tumours. Immunohistochemical analysis of a section of a breast ductal carcinoma specimen stained both with anti-ERBB2 antibody (left) and (the same section) with anti-S100A14 antibody (right). Photographs were taken with a 40x objective.

Oestrogen receptor-α (ER-α) negativity was also significantly associated with elevated levels of S100A14 (p = 0.024). 87.5% (21/24) of ER-α-negative tumour specimens were S100A14-positive. Moreover, a significant association to increased tumour cells proliferation index (MIB) was observed (p = 0.033). 100% (11/11) of tumours assessed as highly proliferating (60-100% of actively proliferating cells) stained positively for S100A14.

Also in this case the subcellular distribution of S100A14 was related to S100A14 protein abundance. 79.7% (59/74) of S100A14-overexpressing tumours displayed prominent plasma membrane and cytoplasmic localization of S100A14 (p < 0.01) relative to 34.8% (8/23) of low-expressing tumours showing predominantly cytoplasmic staining (65.2%; 15/23). In contrast to this tumour cell plasma membrane localization, normal human breast epithelial cells showed solely diffuse cytosolic S100A14 expression (Fig. 11). For the statistical analysis of subcellular localization of S100A14 we omitted the S100A14-negative samples. These results are summarized in Table 6 .

Fig.   11 S100A14 localizes to plasma membrane in breast cancer tissue. S100A14 immunostaining in sections of normal breast ductal epithelial cells (left) and breast ductal carcinoma sections (right). Panels show high power microscopy (magnification, 40x), demonstrating cytoplasmic staining of S100A14 in normal breast ductal epithelial cells (left, indicated by an arrow) and plasma membrane-associated staining of S100A14 in breast carcinoma cells (right).

Our transcriptional and protein analysis demonstrated that expression of S100A14 in primary tumours differed from that in cell line model; the gene was significantly overexpressed in multiple lung and breast tumour samples. Nevertheless, our data also confirmed the differential expression of the gene in a wide range of primary tumours as well as its potential clinical relevance with regard to the positive correlation with ERBB2 overexpression in breast tumours. Therefore, we decided to further characterize the gene and the molecular determinants of its regulation.

Table  6 Association of S100A14 protein expression in breast tumours with clinicopathological factors

To determine if the absence of S100A14 in the vast majority of cultured tumour cell lines could be due to differences between the cell model system and the primary tissue, we examined 9 mouse xenograft tumours of human cultured lung cancer cell lines. They represented the following histopathological types: SCLC (CPC-N, H526, H446, H82, H209, N417), brain metastasis of SCLC (Colo 668), adenocarcinoma (D54), and LCLC (D97). These cell lines do not express endogenous S100A14 in Northern blot analysis.

RT-PCR analysis did not reveal re-expression of the S100A14 transcript in the majority of the examined xenografts ( Fig. 12 ).

Fig.   12 S100A14 is not re-expressed in lung cancer cell line xenografts from nude mice. Nine S100A14-negative human lung cancer cell lines (CPC-N, H526, H446, H82, H209, N417, Colo 668, D54, and D97) were transplanted subcutaneously into immunodefficient mice. Mouse xenograft tumours (MT) and the respective lung cancer cell lines (CL) were examined by RT-PCR. GAPDH was used as a loading control.

An enhanced S100A14 mRNA expression was clearly present only in the D54 xenograft tumour as compared with the corresponding cell line. A very weak mRNA expression was detected in the H82 cell line and the corresponding xenograft as well as in the D97 cell line. A modest mRNA expression was observed in the H209 cell line and a weak expression in the corresponding xenograft.

The major downstream signalling pathways triggered by activated ERBB receptors are ERK1/2 mitogen-activated protein kinase (MAPK), stress-activated protein kinase/c-JUN NH₂-terminal kinase (SAPK/JNK), p38 MAPK, and phosphatidylinositol 3’-kinase (PI3K) pathways. To assess which signalling pathway(s) mediated the stimulatory effect of EGF on S100A14 we applied several pharmacological agents to inhibit these pathways.

First, kinetics of the growth factor-induced response was determined for the three MAPK signalling pathways (ERK1/2, p38, JNK) and the PI3K cascade in order to establish the stimulation efficacy and the optimal treatment time. Immunoblotting with the respective antiphospho antibodies revealed that growth factor stimulation caused a significant increase in ERK1/2 activation which remained on a steady level over a period of 120 min ( Fig. 30 A). The p38 signalling pathway was also markedly activated reaching a maximal induction level between 5 and 30 min after treatment with levels returning to basal by 120 min ( Fig. 30 B). Mitogen exposure also enhanced the phoshorylation and activation of the JNK MAPK cascade in these cells with maximal response at 5 to 15 min after stimulation ( Fig. 30 C). Remarkably, growth factor treatment had no effect on the activation of AKT suggestive of a constitutive activation of the PI3K pathway in these cells ( Fig. 30 D).

Fig.   30 Kinetics of the growth factor-induced response for the ERK1/2, p38, and JNK MAPK pathways and the PI3K cascade. 9442 cells were grown for 3 days in regular culture medium. The medium was then replaced with the medium that was supplemented with a double set of growth factors (referred to as “stimulation medium”; MS). The cells were cultured for the indicated times and harvested. 20 µg of whole protein extracts were subjected to SDS-PAGE followed by Western blot analysis using A: anti-phospho ERK1/2, B: anti-phospho p38, C: anti-phospho SAPK/JNK, and D: anti-phospho AKT antibodies. β-actin, p38, SAPK/JNK, and AKT were used as loading controls, respectively.

Next, we examined the inhibitory efficacy of various pharmacological agents on activation of the intracellular signalling pathways triggered in response to EGF. U0126 (MEK1 and MEK2 inhibitor) markedly suppressed growth factors-induced ERK1/2 activation in 9442 cells ( Fig. 31 A). AG1478 (ERBB receptor tyrosine kinase inhibitor) also markedly attenuated ERK1/2 phosphorylation ( Fig. 31 B). The suppressive efficacy of SB203580 (p38 kinase inhibitor) on the p38 MAPK pathway and SP600125 (JNK kinase inhibitor) on the JNK pathway was confirmed by the ability to block the activation of their downstream targets: HSP27 and cJUN, respectively. This was determined by immunoblotting with the respective antiphospho antibodies ( Fig. 31 C, D). Pretreatment with LY294002 (a PI3K inhibitor) only partially inhibited the response to growth factors ( Fig. 31 E). Increasing concentration of the inhibitor failed to alter the inhibitory response in these cells.

Fig.   31 The inhibitory efficacy of various pharmacological agents on activation of the ERK1/2, p38, and JNK MAPK pathways and the PI3K cascade9442 cells were grown in regular medium for 3 days and then preincubated for 1 hour with A: 20 µM U0126, B: 10 µM AG1478, C: 40 µM SB203580, D: 40 µM SP600125, and E: 40 µM LY294002, respectively, without medium change. Next, the cells were stimulated with the medium that was supplemented with a double set of growth factors (referred as “stimulation medium”; MS) for 10 min (A), 10 min (B), 5 min (C), 5 min (D), and 10 min (E). As negative controls, cells were treated with the vehicle: DMSO (A, B, C, D) or ethanol (E). 20 µg of whole protein extracts were subjected to SDS-PAGE followed by Western blot analysis using anti-phospho ERK1/2 (A, B), anti-phospho HSP27 (C), anti-phospho c-JUN (D), and anti-phospho AKT (E) antibodies. β-actin, p38, c-JUN, and AKT were assayed as loading controls.

To delineate the pathways involved in S100A14 mRNA induction we treated 9442 cells with the MAPK and the PI3K pathway inhibitors prior to EGF stimulation. Treatment with LY294002 does not abrogate growth factor-dependent induction of S100A14 mRNA ( Fig. 32 A). Its expression was also unaffected by SB203580 and SP600125 inhibitors. In contrast with this result, U0126 completely abrogated EGF-induced S100A14 mRNA expression at the time of optimal gene induction (after 12 hours) and this treatment reduced mRNA levels to those seen in unstimulated cells ( Fig. 32 B). To examine whether the intact tyrosine kinase activity of the EGF receptor was necessary for the EGF-induced S100A14 expression, cells were preincubated with AG1478. AG1478 completely prevented EGF-induced S100A14 up-regulation ( Fig. 32 B). None of the concentrations of the inhibitors that were used in this study caused cell detachment or cell death.

Fig.   32 ERK1/2 MAPK signalling pathway determines S100A14 induction following stimulation with EGF9442 cells were grown in regular medium for 3 days and then preincubated for 1 hour with A: 40 µM SP600125, 40 µM SB203580, 40 µM LY294002, B: 20 µM U0126, and 10 µM AG1478, respectively, without medium change. As negative controls, cells were treated with the vehicle: DMSO or ethanol. Next, the cells were stimulated with 50 ng/ml of EGF for 12 hours and harvested. 10 µg of total RNA was size-fractionated followed by Northern blot analysis using ³²Plabelled S100A14 cDNA probe for the detection. 18S RNA was used to correct for equal loading.

These results demonstrated that ERK1/2 MAPK signalling plays a prominent role in regulation of S100A14 transcript in response to growth factors.

No inhibition of S100A14 protein was detected following treatment with the U0126 and AG1478 inhibitors (data not shown). Experiments included both whole cell lysates as well as subcellular fractionated lysates (membrane and soluble fraction). Similarly, no shift in the S100A14 subcellular localization was observed and S100A14 remained compartmentalized to crude membrane and soluble fractions upon induction by EGF, as determined by Western blotting with anti-S100A14 antibody (data not shown).

To further investigate the mechanisms of EGF-mediated S100A14 transcriptional activation, we employed cycloheximide (CHX) – an inhibitor of protein synthesis. Pretreatment of cells with CHX led to a partial reduction in the EGF-induced S100A14 mRNA levels in a dose-dependent manner ( Fig. 33 ). Based on this result, we concluded that the induction of S100A14 following EGF treatment requires a new protein synthesis. Therefore, S100A14 is likely to be an indirect transcriptional target of EGF signalling and it is conceivable that the synthesis of other protein(s) is involved in its transcriptional activation.

Fig.   33 EGF-induced S100A14 expression is dependent on de novo protein synthesis. 9442 cells were grown in regular medium for 3 days and then preincubated for 1 hour with 2, 5, and 10 µg/ml of cycloheximide (CHX), respectively, without medium change. As negative controls, cells were treated with the vehicle DMSO. Next, the cells were stimulated with 50 ng/ml EGF for 12 hours and harvested. 10 µg of total RNA was size-fractionated followed by Northern blot analysis using ³²Plabelled S100A14 cDNA probe for the detection. 18S RNA was used as a loading control.