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Induction of apoptosis of metastatic mammary carcinoma cells in vivo by disruption of tumor cell surface CD44 function.

Yu Q, Toole BP, Stamenkovic I - J. Exp. Med. (1997)

Bottom Line: To understand how the hyaluronan receptor CD44 regulates tumor metastasis, the murine mammary carcinoma TA3/St, which constitutively expresses cell surface CD44, was transfected with cDNAs encoding soluble isoforms of CD44 and the transfectants (TA3sCD44) were compared with parental cells (transfected with expression vector only) for growth in vivo and in vitro.However, although parental cells were dividing and forming clusters within lung tissue 48 h following injection, >80% of TA3sCD44 cells underwent apoptosis.Although sCD44 transfectants displayed a marked reduction in their ability to internalize and degrade hyaluronan, they elicited abundant local hyaluronan production within invaded lung tissue, comparable to that induced by parental cells.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pathology Unit and MGH Cancer Center, Massachusetts General Hospital, Charlestown Navy Yard, Boston, Massachusetts 02129, USA.

ABSTRACT
To understand how the hyaluronan receptor CD44 regulates tumor metastasis, the murine mammary carcinoma TA3/St, which constitutively expresses cell surface CD44, was transfected with cDNAs encoding soluble isoforms of CD44 and the transfectants (TA3sCD44) were compared with parental cells (transfected with expression vector only) for growth in vivo and in vitro. Local release of soluble CD44 by the transfectants inhibited the ability of endogenous cell surface CD44 to bind and internalize hyaluronan and to mediate TA3 cell invasion of hyaluronan-producing cell monolayers. Mice intravenously injected with parental TA3/St cells developed massive pulmonary metastases within 21-28 d, whereas animals injected with TA3sCD44 cells developed few or no tumors. Tracing of labeled parental and transfectant tumor cells revealed that both cell types initially adhered to pulmonary endothelium and penetrated the interstitial stroma. However, although parental cells were dividing and forming clusters within lung tissue 48 h following injection, >80% of TA3sCD44 cells underwent apoptosis. Although sCD44 transfectants displayed a marked reduction in their ability to internalize and degrade hyaluronan, they elicited abundant local hyaluronan production within invaded lung tissue, comparable to that induced by parental cells. These observations provide direct evidence that cell surface CD44 function promotes tumor cell survival in invaded tissue and that its suppression can induce apoptosis of the invading tumor cells, possibly as a result of impairing their ability to penetrate the host tissue hyaluronan barrier.

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(A) [3H]-HA uptake and degradation. Soluble CD44 expression by the transfected TA3 cells results in a reduction of up to three-fold  of 3H-HA uptake and degradation. (B) Invasion of G8 myoblast monolayers  by TA3neo and TA3sCD44 cells. TA3neo (a and c) and TA3sCD44v6-10  (b and d) cells (5 × 103 cells/well) were seeded onto DMSO-fixed G8  monolayers which were untreated (a and b) or treated with streptomyces  hyaluronidase (10 U/ml, ICN, Costa Mesa, CA; c and d) at 37°C for 3 h. 6 d  after seeding of TA3 transfectants, TA3 neo cells are observed to penetrate  and invade the monolayer, treated or not with hyaluronidase, and form tumor cell islets (arrows, TA3 neo cells). By contrast, TA3sCD44 cells were  unable to penetrate the untreated G8 monolayer (b, arrowheads), whereas  treatment of the fixed G8 cells with hyaluronidase restored the ability of  TA3sCD44 cell to penetrate the monolayer (d, open arrows).
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Figure 4: (A) [3H]-HA uptake and degradation. Soluble CD44 expression by the transfected TA3 cells results in a reduction of up to three-fold of 3H-HA uptake and degradation. (B) Invasion of G8 myoblast monolayers by TA3neo and TA3sCD44 cells. TA3neo (a and c) and TA3sCD44v6-10 (b and d) cells (5 × 103 cells/well) were seeded onto DMSO-fixed G8 monolayers which were untreated (a and b) or treated with streptomyces hyaluronidase (10 U/ml, ICN, Costa Mesa, CA; c and d) at 37°C for 3 h. 6 d after seeding of TA3 transfectants, TA3 neo cells are observed to penetrate and invade the monolayer, treated or not with hyaluronidase, and form tumor cell islets (arrows, TA3 neo cells). By contrast, TA3sCD44 cells were unable to penetrate the untreated G8 monolayer (b, arrowheads), whereas treatment of the fixed G8 cells with hyaluronidase restored the ability of TA3sCD44 cell to penetrate the monolayer (d, open arrows).

Mentions: To determine whether the reduction of TA3sCD44 cell ability to form metastases correlates with the suppression of known functions of cell surface CD44 by locally secreted CD44, TA3sCD44 transfectants were compared to parental cells for HA binding and internalization, invasion of cell monolayers, response to growth factors, and adhesion to a panel of ECM proteins. TA3neo and TA3sCD44 cells responded comparably to basic fibroblast growth factor (FGF) and heparin-binding epidermal growth factor (EGF), both of which bound v3-containing CD44 isoforms (15), and displayed similar adhesion to laminin, collagen type IV, and fibronectin in vitro (data not shown). By contrast, expression of soluble CD44 in TA3 cells, but not soluble ICAM-1 or CD44R43A, was found to reduce their ability to attach to HA substrata (Table 1). In addition, TA3sCD44 cells displayed a decreased ability to internalize radiolabeled HA (Fig. 4 A) and invade G8 myoblast monolayers, which produce abundant pericellular HA (Fig. 4 B and data not shown). The ability of TA3sCD44 transfectants to penetrate the G8 monolayers was restored after treatment of the monolayers with streptomyces HA (Fig. 4 B), indicating that pericellular HA may constitute a barrier to tissue invasion by cells whose CD44-dependent ability to bind and internalize HA is compromised. Treatment of G8 monolayers with heparinase, used as a control, could not restore TA3sCD44 cell invasiveness (data not shown).


Induction of apoptosis of metastatic mammary carcinoma cells in vivo by disruption of tumor cell surface CD44 function.

Yu Q, Toole BP, Stamenkovic I - J. Exp. Med. (1997)

(A) [3H]-HA uptake and degradation. Soluble CD44 expression by the transfected TA3 cells results in a reduction of up to three-fold  of 3H-HA uptake and degradation. (B) Invasion of G8 myoblast monolayers  by TA3neo and TA3sCD44 cells. TA3neo (a and c) and TA3sCD44v6-10  (b and d) cells (5 × 103 cells/well) were seeded onto DMSO-fixed G8  monolayers which were untreated (a and b) or treated with streptomyces  hyaluronidase (10 U/ml, ICN, Costa Mesa, CA; c and d) at 37°C for 3 h. 6 d  after seeding of TA3 transfectants, TA3 neo cells are observed to penetrate  and invade the monolayer, treated or not with hyaluronidase, and form tumor cell islets (arrows, TA3 neo cells). By contrast, TA3sCD44 cells were  unable to penetrate the untreated G8 monolayer (b, arrowheads), whereas  treatment of the fixed G8 cells with hyaluronidase restored the ability of  TA3sCD44 cell to penetrate the monolayer (d, open arrows).
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Figure 4: (A) [3H]-HA uptake and degradation. Soluble CD44 expression by the transfected TA3 cells results in a reduction of up to three-fold of 3H-HA uptake and degradation. (B) Invasion of G8 myoblast monolayers by TA3neo and TA3sCD44 cells. TA3neo (a and c) and TA3sCD44v6-10 (b and d) cells (5 × 103 cells/well) were seeded onto DMSO-fixed G8 monolayers which were untreated (a and b) or treated with streptomyces hyaluronidase (10 U/ml, ICN, Costa Mesa, CA; c and d) at 37°C for 3 h. 6 d after seeding of TA3 transfectants, TA3 neo cells are observed to penetrate and invade the monolayer, treated or not with hyaluronidase, and form tumor cell islets (arrows, TA3 neo cells). By contrast, TA3sCD44 cells were unable to penetrate the untreated G8 monolayer (b, arrowheads), whereas treatment of the fixed G8 cells with hyaluronidase restored the ability of TA3sCD44 cell to penetrate the monolayer (d, open arrows).
Mentions: To determine whether the reduction of TA3sCD44 cell ability to form metastases correlates with the suppression of known functions of cell surface CD44 by locally secreted CD44, TA3sCD44 transfectants were compared to parental cells for HA binding and internalization, invasion of cell monolayers, response to growth factors, and adhesion to a panel of ECM proteins. TA3neo and TA3sCD44 cells responded comparably to basic fibroblast growth factor (FGF) and heparin-binding epidermal growth factor (EGF), both of which bound v3-containing CD44 isoforms (15), and displayed similar adhesion to laminin, collagen type IV, and fibronectin in vitro (data not shown). By contrast, expression of soluble CD44 in TA3 cells, but not soluble ICAM-1 or CD44R43A, was found to reduce their ability to attach to HA substrata (Table 1). In addition, TA3sCD44 cells displayed a decreased ability to internalize radiolabeled HA (Fig. 4 A) and invade G8 myoblast monolayers, which produce abundant pericellular HA (Fig. 4 B and data not shown). The ability of TA3sCD44 transfectants to penetrate the G8 monolayers was restored after treatment of the monolayers with streptomyces HA (Fig. 4 B), indicating that pericellular HA may constitute a barrier to tissue invasion by cells whose CD44-dependent ability to bind and internalize HA is compromised. Treatment of G8 monolayers with heparinase, used as a control, could not restore TA3sCD44 cell invasiveness (data not shown).

Bottom Line: To understand how the hyaluronan receptor CD44 regulates tumor metastasis, the murine mammary carcinoma TA3/St, which constitutively expresses cell surface CD44, was transfected with cDNAs encoding soluble isoforms of CD44 and the transfectants (TA3sCD44) were compared with parental cells (transfected with expression vector only) for growth in vivo and in vitro.However, although parental cells were dividing and forming clusters within lung tissue 48 h following injection, >80% of TA3sCD44 cells underwent apoptosis.Although sCD44 transfectants displayed a marked reduction in their ability to internalize and degrade hyaluronan, they elicited abundant local hyaluronan production within invaded lung tissue, comparable to that induced by parental cells.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pathology Unit and MGH Cancer Center, Massachusetts General Hospital, Charlestown Navy Yard, Boston, Massachusetts 02129, USA.

ABSTRACT
To understand how the hyaluronan receptor CD44 regulates tumor metastasis, the murine mammary carcinoma TA3/St, which constitutively expresses cell surface CD44, was transfected with cDNAs encoding soluble isoforms of CD44 and the transfectants (TA3sCD44) were compared with parental cells (transfected with expression vector only) for growth in vivo and in vitro. Local release of soluble CD44 by the transfectants inhibited the ability of endogenous cell surface CD44 to bind and internalize hyaluronan and to mediate TA3 cell invasion of hyaluronan-producing cell monolayers. Mice intravenously injected with parental TA3/St cells developed massive pulmonary metastases within 21-28 d, whereas animals injected with TA3sCD44 cells developed few or no tumors. Tracing of labeled parental and transfectant tumor cells revealed that both cell types initially adhered to pulmonary endothelium and penetrated the interstitial stroma. However, although parental cells were dividing and forming clusters within lung tissue 48 h following injection, >80% of TA3sCD44 cells underwent apoptosis. Although sCD44 transfectants displayed a marked reduction in their ability to internalize and degrade hyaluronan, they elicited abundant local hyaluronan production within invaded lung tissue, comparable to that induced by parental cells. These observations provide direct evidence that cell surface CD44 function promotes tumor cell survival in invaded tissue and that its suppression can induce apoptosis of the invading tumor cells, possibly as a result of impairing their ability to penetrate the host tissue hyaluronan barrier.

Show MeSH
Related in: MedlinePlus