HAb18G/CD147 cell-cell contacts confer resistance of a HEK293 subpopulation to anoikis in an E-cadherin-dependent manner.

Abstract

ABSTRACT: BACKGROUND: Acquisition of resistance to "anoikis" facilitates the survival of cells under independent matrix-deficient conditions, such as cells in tumor progression and the production of suspension culture cells for biomedical engineering. There is evidence suggesting that CD147, an adhesion molecule associated with survival of cells in tumor metastasis and cell-cell contacts, plays an important role in resistance to anoikis. However, information regarding the functions of CD147 in mediating cell-cell contacts and anoikis-resistance remains limited and even self-contradictory. RESULTS: An anoikis-resistant clone (HEK293ar), derived from anoikis-sensitive parental Human Embryonic Kidney 293 cells, survived anoikis by the formation of cell-cell contacts. The expression of HAb18G/CD147 (a member of the CD147 family) was upregulated and the protein was located at cell-cell junctions. Upregulation of HAb18G/CD147 in suspended HEK293ar cells suppressed anoikis by mediating the formation of cell-cell adhesions. Anoikis resistance in HEK293ar cells also required E-cadherin-mediated cell-cell contacts. Knock-down of HAb18G/CD147 and E-cadherin inhibited cell-cell contacts formation and increased anoikis sensitivity respectively. When HAb18G/CD147 was downregulated, E-cadherin expression in HEK293ar cells was significantly suppressed; however, knockdown of E-cadherin by E-cadherin siRNA or blocking of E-cadherin binding activity with a specific antibody and EDTA had no significant effect on HAb18G/CD147 expression. Finally, pretreatment with LY294002, a phosphoinositide 3-kinase (PI3K/AKT) inhibitor, disrupted cell-cell contacts and decreased cell number, but this was not the case in cells treated with the extracellular signal-regulated kinase (ERK) inhibitor PD98059. CONCLUSIONS: Our results provide new evidence that HAb18G/CD147-mediated cell-cell contact confers anoikis resistance in an E-cadherin-dependent manner; and cell-cell contact mediated resistance to anoikis implicates PI3K pathway in a highly relevant cell model (HEK293ar). Understanding of the role of HAb18G/CD147 cell-cell contacts in anoikis resistance may help in understanding the survival of cells in anchorage-independent growth, such as cells in tumor metastasis and suspension culture produced for biomedical engineering. Our results also contribute to a better understanding of the biology of HEK293 cell spheroids, a major workhorse for producing human therapeutic agents and viral vaccines.

Full text

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Ma et al. BMC Cell Biology 2010, 11:27
http://www.biomedcentral.com/1471-2121/11/27
Open Access
RESEARCH ARTICLE
HAb18G/CD147 cell-cell contacts confer resistance
of a HEK293 subpopulation to anoikis in an
E-cadherin-dependent manner
BioMed Central
© 2010 Ma et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons At-
tribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly cited.
Research article
Xiao-Kui Ma†1, Li Wang†1, Yu Li1, Xiang-Ming Yang1, Pu Zhao1, HaoTang1, Ping Zhu2, Ling Li*1 and Zhi-Nan Chen*1
Abstract
Background: Acquisition of resistance to "anoikis" facilitates the survival of cells under independent matrix-deficient
conditions, such as cells in tumor progression and the production of suspension culture cells for biomedical
engineering. There is evidence suggesting that CD147, an adhesion molecule associated with survival of cells in tumor
metastasis and cell-cell contacts, plays an important role in resistance to anoikis. However, information regarding the
functions of CD147 in mediating cell-cell contacts and anoikis-resistance remains limited and even self-contradictory.
Results: An anoikis-resistant clone (HEK293ar), derived from anoikis-sensitive parental Human Embryonic Kidney 293
cells, survived anoikis by the formation of cell-cell contacts. The expression of HAb18G/CD147 (a member of the CD147
family) was upregulated and the protein was located at cell-cell junctions. Upregulation of HAb18G/CD147 in
suspended HEK293ar cells suppressed anoikis by mediating the formation of cell-cell adhesions. Anoikis resistance in
HEK293ar cells also required E-cadherin-mediated cell-cell contacts. Knock-down of HAb18G/CD147 and E-cadherin
inhibited cell-cell contacts formation and increased anoikis sensitivity respectively. When HAb18G/CD147 was
downregulated, E-cadherin expression in HEK293ar cells was significantly suppressed; however, knockdown of E-
cadherin by E-cadherin siRNA or blocking of E-cadherin binding activity with a specific antibody and EDTA had no
significant effect on HAb18G/CD147 expression. Finally, pretreatment with LY294002, a phosphoinositide 3-kinase
(PI3K/AKT) inhibitor, disrupted cell-cell contacts and decreased cell number, but this was not the case in cells treated
with the extracellular signal-regulated kinase (ERK) inhibitor PD98059.
Conclusions: Our results provide new evidence that HAb18G/CD147-mediated cell-cell contact confers anoikis
resistance in an E-cadherin-dependent manner; and cell-cell contact mediated resistance to anoikis implicates PI3K
pathway in a highly relevant cell model (HEK293ar). Understanding of the role of HAb18G/CD147 cell-cell contacts in
anoikis resistance may help in understanding the survival of cells in anchorage-independent growth, such as cells in
tumor metastasis and suspension culture produced for biomedical engineering. Our results also contribute to a better
understanding of the biology of HEK293 cell spheroids, a major workhorse for producing human therapeutic agents
and viral vaccines.
Background
CD147, an extracellular matrix metalloproteinase inducer
(also known as EMMPRIN, basigin, M6), is a plasma
membrane-bound glycoprotein that functions as an
adhesion molecule. It is expressed at high levels on a vari-
ety of malignant human cancers and some immortalized
cell lines. Our laboratory previously identified a novel
hepatoma associated antigen named HAb18G, which was
obtained by cloning a human hepato-cellular carcinoma
(HCC) cDNA library and screening with the anti-hepa-
toma monoclonal antibody HAb18 [1]. The nucleotide
acid and amino acid sequences of HAb18G are identical
* Correspondence: Liling25@fmmu.edu.cn
, Zhinanchen@fmmu.edu.cn
Cell Engineering Research Center & Department of Cell Biology, National Key
Discipline of Cell Biology, State Key Laboratory of Cancer Biology, Fourth
Military Medical University, 17 Changle West Road, Xi'an 710032, PR China
† Contributed equally
Full list of author information is available at the end of the article

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to those of CD147 [2]. HAb18G/CD147 was highly
expressed by HCC cells and tissues, and increased
HAb18G/CD147 expression stimulated both the growth
and invasiveness of HCC cells, much as CD147 functions
in other cancer cells [3-5].
The acquisition of resistance to anoikis, a form of apop-
tosis triggered by loss or alteration of cell-cell or cell-
matrix anchorage, is critical for the survival of cells in
tumor progression and suspension growth used in engi-
neering. Resistance to anoikis is emerging as a hallmark
of metastatic cancer cells, important in tumor progres-
sion because it increases survival times in the absence of
cell anchorage, facilitating migration and reattachment,
and therefore increasing the probability of metastasis [6].
Furthermore, acquisition of anoikis resistance is required
for cells used in engineering during adaptation to suspen-
sion culture and spheroid growth.
More recently, CD147 has been reported as an anoikis
suppressor, promoting anchorage-independent growth
by stimulating hyaluronan production [7] and regulating
the anoikis signal pathway by upregulating Bim [4]. How-
ever, it is not clear whether the role of CD147 in anoikis
resistance is related to cell adhesion, which is a basic
function of this molecule in addition to its role in stimu-
lating matrix metalloproteinase (MMP) secretion [8].
Different bioactive epitopes of CD147 involved in regu-
lating cell adhesion have been identified [9]. CD147 has
also been reported to participate in forming compacted
cell aggregates by regulating fibronectin matrix assembly
[4] and cell-cell adhesion [10]. The binding of CD147
mAb to CD147 may mimic natural ligand-receptor bind-
ing and induce homotypic U937 monocytic cell aggrega-
tion via the LFA-1/ICAM-1 pathway [11]. In contrast,
Cho reported that antibodies to CD147 are potent inhibi-
tors of homotypic U937 aggregation induced via CD98
ligation [12]. Because the establishment/maintenance of
cell-cell contacts is considered an important environmen-
tal condition for physiological resistance to anoikis, we
hypothesized that CD147 may confer anoikis resistance
by mediating cell-cell adhesion. Unfortunately, direct evi-
dence for the role of CD147 in mediating cell-cell con-
tacts and anoikis resistance is very limited and even self-
contradictory. Furthermore, it is not clear whether
CD147 is directly involved in cell adhesion either as an
adhesion signal transmitting molecule or a regulator.
The aim here was to explore whether HAb18G/CD147
is involved in forming cell-cell contacts, and whether this
contributed to its role in regulating anoikis resistance. A
transformed cell line, Human Embryonic Kidney (HEK)
293, was chosen as the model because our previous
results confirmed that these cells express HAb18G/
CD147 [13]. We also acquired an anoikis-resistant sub-
population (HEK293ar) from the anoikis-sensitive paren-
tal HEK293 cells. Together, these two cell types provide
an ideal model for exploring the role of HAb18G/CD147
as a cell-cell adhesion molecule preventing anoikis. Our
results show that HAb18G/CD147 cell-cell contacts con-
fer anoikis resistance and this function also involves E-
cadherin expression in HEK293ar cell spheroids.
Results
Anoikis-resistant HEK293ar cells survive anoikis by the
formation of cell-cell contacts
To explore the role of cell-cell contacts in preventing
anoikis, we generated a subpopulation (HEK293ar) with
an anoikis-resistant phenotype derived from HEK293
cells using sequential cycles of adhesion and suspension
culture [14]. Upon detachment, the apoptotic ratio in
HEK293ar cells was lower than in the parental cells, as
shown by quantifying the proportions in sub-G1 by flow
cytometry (6.97 ± 3.1% versus 37.3 ± 2.3%, data not
shown). When HEK293ar cells were cultured in suspen-
sion for 1, 10 and 30 days, the mean sub-G1 proportions
were 8.7, 11.7, and 15.1%, respectively, with little change
in the apoptotic ratio (Fig. 1A). Moreover, this subpopu-
lation maintained the anoikis resistance phenotype in
vitro for 6 months (data not shown). Additionally, after
~6-10 h suspension culture, the HEK293ar cells sponta-
neously and gradually formed cell-cell contacts and gen-
erated multi-cellular spheroids that could still grow as
adhesion cultures, whereas the parental HEK293 cells did
not (Fig. 1B). Ultrastructural examination demonstrated
that the suspended spheroids comprised many single
cells. In each spheroid, chromatin masses of moderate
electron density were dispersed in the nuclei, and the
nucleoli were conspicuous (Fig. 1C, arrowed). Cells
within the multi-cellular spheroids adhered laterally to
each other through diverse specialized intercellular junc-
tions (Fig. 1D, arrowhead). Pretreatment with EDTA
solution to disrupt calcium-dependent cell-cell contacts
also disrupted the adhesions and caused massive DNA
fragmentation in HEK293ar cells, as shown by TUNEL
assay (Fig. 1E). Interestingly, single cells detached from
the aggregates gave more intense TUNEL staining (Fig.
1E, arrowhead). In contrast, TUNEL assays and ultra-
structural examination demonstrated that HEK293 cells
clearly underwent anoikis (data not shown). It was previ-
ously reported that cell-cell contacts prevent anoikis in
primary human colonic epithelial cells [15]. The present
results also suggest that adjacent HEK293ar epithelial
cells form cell-cell contacts, suppressing anoikis under
matrix-deficient conditions.
HAb18G/CD147 promotes HEK293ar cell survival in
suspension by mediating cell-cell contacts
We next explored whether HAb18G/CD147 was involved
in HEK293ar cell spheroid survival by mediating cell-cell
contacts. HAb18G/CD147 expression was compared

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between HEK293ar and HEK293 cells in suspension cul-
ture. HEK293ar cells expressed significantly more
HAb18G/CD147 than the parental cells after 24 h (Fig.
2A). Positive immunofluorescence staining for CD147
was restricted to the cell-cell contacts of HEK293ar (Fig.
2B, merge, arrowhead). These results may imply a corre-
lation between HAb18G/CD147expression and cell-cell
contacts-directed anoikis resistance, as the level of
HAb18G/CD147 expression was similar in both cell types
in adhesion culture (data not shown). To obtain further
insight into this correlation, HEK293ar cells were treated
with HAb18G/CD147 siRNA in adhesion culture, and
apoptosis and cell-cell contacts formation were deter-
mined in suspension culture. When HAb18G/CD147
expression was reduced by ~70% (Figs. 2C, D; **p < 0.01),
HEK293ar cells underwent anoikis with a sub-G1 propor-
tion of 37.9 ± 2.1% (Fig. 2E). Cell-cell contacts formation
was totally inhibited after suspension, whereas the con-
trol cells formed multicellular aggregates after as little as
12 h in suspension (Fig. 2F). These results indicate that
HAb18G/CD147 confers anoikis resistance of HEK293ar
cells to anoikis by mediating cell-cell contacts formation.
Figure 1 Anoikis-resistant HEK293ar cells survives anoikis through cell-cell contacts. An anoikis-resistant HEK293 cell subpopulation (HEK293ar)
was obtained by sequential cycles of adhesion and suspension culture. A. Anoikis in HEK293ar cells in suspension culture at different time points. Flow
cytometry (histogram) shows that the apoptosis rate changed little during suspension culture of HEK293ar. The x and y-axes indicate the size of DNA
and the number of cells counted, respectively. FL3-H, a standard term for flow cytometry, represents measurement of the fluorescence intensity of
propidium iodide (PI) at a super-red wavelength (670 nm). The results are means ± S.D. (n = 4-6). B. The morphology of HEK293ar and parental HEK293
cells in suspension and adhesion culture revealed by microscopy (n = 4-10). Magnification: ×200. C. TEM of a multicellular HEK293ar spheroid. Chro-
matin masses of moderate electron density are dispersed in the nuclei and nucleoli are conspicuous (arrowed). The cells were observed in 4-6 inde-
pendent sections. Bar, 2 μm. D. A junctional complex with thickened membranes (arrowed) in suspended HEK293ar cells viewed under SEM. Bar, 50
nm. The cells were viewed in 4-10 independent sections (at least 100 cells/section). E. EDTA (10 mmol/l) treatment disrupted the cell-cell contacts and
resulted in massive apoptosis as determined by TUNEL assay. Arrowheads show that single cells detached from the aggregates give more intense
TUNEL staining. Magnification: ×200. The apoptotic nuclei were counted in 4-10 independent sections (at least 500 nuclei/section).

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Cell-cell contact-directed survival in suspension involves E-
cadherin
Upon detachment, anchorage-independent Ewing sar-
coma cells suppressed anoikis through a pathway involv-
ing E-cadherin-dependent cell-cell adhesion [16]. Thus
we explored whether E-cadherin was also involved in
suppressing anoikis by mediating cell-cell adhesion in
HEK293ar cells. As anticipated, increased E-cadherin
expression was confirmed in HEK293ar cells after 24 h
suspension culture (Figs. 3A, B, *p < 0.05), and positive
staining was partly located around the cell-cell contacts
(Fig. 3C). Also, no significant expression of E-cadherin in
completely suspended parental HEK293 cells or in the
initially suspended HEK293ar cells was found by immun-
ofluorescence. A reasonable explanation could be that E-
cadherin expression decreases dramatically upon cell-cell
detachment [17]. In addition, a 70-80% reduction of E-
cadherin expression by siRNA (Figs. 3D, E; ** p < 0.01), as
determined by western blotting, totally inhibited cell-cell
contacts formation (Fig. 3F) and decreased the degree of
cell aggregation with 70-80%, scored as described in
Methods. Knockdown of E-cadherin led to a marked
increase in the mean sub-G1 proportion and a little
shifted peaks in Flow cytometric histogram (Fig. 3G, * p <
0.05), and it also decreased the cellular DNA content via
fluorescent dye binding determined with a CyQUANT®
Figure 2 HAb18G/CD147 promotes HEK293ar cells survival by mediating cell-cell contacts. A. Western blotting to reveal HAb18G/CD147 ex-
pression in HEK293ar and parental HEK293 cells in suspension culture. Two major forms of HAb18G/CD147 (43-66 and 35 kDa) were analyzed. α-Tu-
bulin was used as a loading control (n = 4-6). B. Immunofluorescence of HAb18G/CD147 under laser scanning confocal microscope (Olympus FV1000,
Tokyo, Japan; n = 4-10). The nuclei were counterstained with DAPI (4',6-diamidino-2-phenylindole). Magnification: ×1200. C. Western blotting to reveal
HAb18G/CD147 expression in HEK293ar cells in HAb18G/CD147 RNA interference (n = 4-6). α-Tubulin was used as a loading control. D. Comparison
of the gray scale ratio of HAb18G/CD147/α-tubulin in HAb18G/CD147 RNA interference (n = 4-6). ** p < 0.01, siRNA versus scrambled. E. Flow cyto-
metric quantification of anoikis after siRNA treatment (n = 4-6). Anoikis was determined as described in Fig. 1A. F. Cell-cell contacts formation with
time after treatment of targeted HAb18G/CD147-siRNA under the phase-contrast microscope (n = 4-6). Magnification: × 400. Each value represents
the mean ± SD of at least triplicate determinations. Results are the representative of three similar experiments.

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Figure 3 Cell-cell contact-directed HEK293ar cell survival requires the involvement of E-cadherin. A. Increased E-cadherin expression in
HEK293ar cells revealed by western blotting (n = 4-6). B. Comparison of the gray scale ratio of E-cadherin/α-tubulin between HEK293 and HEK293ar
cells (n = 4-6). * p < 0.05, HEK293 versus HEK293ar. C. E-cadherin expression in HEK293ar and HEK293 cells revealed by immunofluorescence under
laser scanning confocal microscope (Olympus FV1000, Tokyo, Japan; n = 4-10). Magnification: × 1600. D. Western blotting to reveal E-cadherin expres-
sion in E-cadherin RNA interference (n = 4-6). E. Comparison of the gray scale ratio of E-cadherin/α-tubulin in E-cadherin RNA interference (n = 4-6).
** p < 0.01, siRNA versus scrambled. F. The effect of E-cadherin RNA interference on the cell-cell contacts formation. Magnification: × 1000. The nuclei
were counterstained with DAPI. The cells were viewed in 4-6 independent sections (at least 300 cells/section). G. Flow cytometric quantification of
anoikis after E-cadherin siRNA-treatment (n = 4-6). H. The effect of E-cadherein knockdown on the cell number of HEK293ar in suspension. * p < 0.05,
siRNA versus scrambled. The quantification of relative cell number was done with CyQUANT® NF Cell Proliferation Assay Kit. This kit measures the cel-
lular DNA content via fluorescent dye binding, which is closely proportional to cell number (n = 4-10). Each value represents the mean ± SD of triplicate
determinations. Results are the representative of three similar experiments.