**4. Summary, therapeutic strategies and future research to elucidate fibrin(ogen)-mediated mechanisms of breast cancer metastasis**

#### **4.1 Summary and therapeutic strategy using free peptide 15-42 to inhibit breast cancer metastasis as depicted in Fig. 10, Steps 1-11**

Regardless of the subtype of breast cancer, once the primary tumor becomes established (*Step 1*), it needs to develop its own blood supply for nutrient delivery and removal of toxic waste (*Step 2*). Breast cancer cells produce VEGF, which initiates permeability of nearby blood vessels allowing plasma Fg to leak into the tumor stroma promoting desmoplasia and deposition of a provisional fibrin(ogen) matrix in the tumor microenvironment (*Step 2*). Alternatively, endogenous synthesis of Fg by breast cancer cells could induce cancer progression. Thus, the innate immune response is activated to defend the host against this neoplastic insult. Release of IL-6 systemically leads to increased production of plasma Fg and fibrin formation resulting in exposure of 15-42 and binding to VE-cadherin, a step critical for angiogenesis (Bach et al., 1998b; Martinez et al., 2001). Furthermore, VEGF binds to Fg and fibrin with high affinity (Sahni & Francis, 2000), which may be necessary for Fg to enhance VEGF-mediated endothelial cell permeability without potentiating endothelial cell proliferation. In contrast, VE-cadherin and VEGF receptor-2 form a signaling complex to promote endothelial cell proliferation (Carmeliet et al., 1999; Dejana, 2004; Esser et al., 1998). Fibrin(ogen) potentiates FGF-2- but not VEGF-induced proliferation of endothelial cells, angiogenesis and cancer cell growth (Rybarczyk & Simpson-Haidaris, 2000; Sahni & Francis, 2000; Sahni et al., 2006; Sahni et al., 2008; Sahni et al., 1999; Simpson-Haidaris, 1997; Simpson-Haidaris & Rybarczyk, 2001). Furthermore, fibrin(ogen) enhances cell migration and cancer invasion through tumor stroma, and TEM, *i.e*., intravasation of breast cancer cells into the blood stream (*Step 3*) (Roche et al., 2003; Rybarczyk et al., 2003; Sahni et al., 2009). Fg and fibrin can bridge between cells of the same or different kinds (Kloczewiak et al., 1983; Languino et al., 1995; Languino et al., 1993; Saito et al., 2002; Sriramarao et al., 1996) and form aggregates or tumor emboli coated with fibrin(ogen) (*Step 4*). Because the host immune system does not recognize fibrin(ogen)-coated tumor emboli (Palumbo et al., 2005), immune-mediated destruction of tumor cells does not occur and these tumor emboli travel through the circulation to sites favorable for metastatic growth (*Steps 5 & 6*) such as lung. To establish metastatic growth, tumor emboli need to leave the circulation and enter lung tissue (*Steps 7 and 8*) where they find a receptive niche (*Step 9*) to begin the process again. Tumor

The data indicate that 10 g/ml (30 nM) Fg enhanced the flux of FITC-dextran to the bottom chamber of the transwell plate at low doses of VEGF (0.05 and 0.1 ng/ml); however, the additive effect on induction of endothelial cell permeability was lost at 0.5 ng/ml and higher concentrations of VEGF (**Fig. 9**). Fg-enhancement of VEGF-induced permeability is rapid and saturated within 5 min, whereas 5 ng/ml of VEGF is required to induce a similar amount of FITC-dextran flux as 30 nM Fg + 0.05 ng/ml, *i.e.*, 100-fold less VEGF. Studies by others suggest that low-dose VEGF mediates inflammation to promote cell survival of vascular and nonvascular cells such as those of the CNS, prior to induction of angiogenesis (Abumiya et al., 2005; Croll et al., 2004). Furthermore, VEGF colocalizes with exuded Fg at sites of edema in renal cell carcinoma (Verheul et al., 2010). Together with the aforementioned published data, our results suggest that Fg may regulate vascular permeability induced by low doses of VEGF without inducing EC proliferation—such a

response would be conducive to fibrinogen induction of breast cancer cell TEM*.*

**4. Summary, therapeutic strategies and future research to elucidate fibrin(ogen)-mediated mechanisms of breast cancer metastasis** 

**metastasis as depicted in Fig. 10, Steps 1-11** 

**4.1 Summary and therapeutic strategy using free peptide 15-42 to inhibit breast cancer** 

Regardless of the subtype of breast cancer, once the primary tumor becomes established (*Step 1*), it needs to develop its own blood supply for nutrient delivery and removal of toxic waste (*Step 2*). Breast cancer cells produce VEGF, which initiates permeability of nearby blood vessels allowing plasma Fg to leak into the tumor stroma promoting desmoplasia and deposition of a provisional fibrin(ogen) matrix in the tumor microenvironment (*Step 2*). Alternatively, endogenous synthesis of Fg by breast cancer cells could induce cancer progression. Thus, the innate immune response is activated to defend the host against this neoplastic insult. Release of IL-6 systemically leads to increased production of plasma Fg and fibrin formation resulting in exposure of 15-42 and binding to VE-cadherin, a step critical for angiogenesis (Bach et al., 1998b; Martinez et al., 2001). Furthermore, VEGF binds to Fg and fibrin with high affinity (Sahni & Francis, 2000), which may be necessary for Fg to enhance VEGF-mediated endothelial cell permeability without potentiating endothelial cell proliferation. In contrast, VE-cadherin and VEGF receptor-2 form a signaling complex to promote endothelial cell proliferation (Carmeliet et al., 1999; Dejana, 2004; Esser et al., 1998). Fibrin(ogen) potentiates FGF-2- but not VEGF-induced proliferation of endothelial cells, angiogenesis and cancer cell growth (Rybarczyk & Simpson-Haidaris, 2000; Sahni & Francis, 2000; Sahni et al., 2006; Sahni et al., 2008; Sahni et al., 1999; Simpson-Haidaris, 1997; Simpson-Haidaris & Rybarczyk, 2001). Furthermore, fibrin(ogen) enhances cell migration and cancer invasion through tumor stroma, and TEM, *i.e*., intravasation of breast cancer cells into the blood stream (*Step 3*) (Roche et al., 2003; Rybarczyk et al., 2003; Sahni et al., 2009). Fg and fibrin can bridge between cells of the same or different kinds (Kloczewiak et al., 1983; Languino et al., 1995; Languino et al., 1993; Saito et al., 2002; Sriramarao et al., 1996) and form aggregates or tumor emboli coated with fibrin(ogen) (*Step 4*). Because the host immune system does not recognize fibrin(ogen)-coated tumor emboli (Palumbo et al., 2005), immune-mediated destruction of tumor cells does not occur and these tumor emboli travel through the circulation to sites favorable for metastatic growth (*Steps 5 & 6*) such as lung. To establish metastatic growth, tumor emboli need to leave the circulation and enter lung tissue (*Steps 7 and 8*) where they find a receptive niche (*Step 9*) to begin the process again. Tumor cell proliferation and angiogenesis (*Step 10*) in lung results in metastatic disease (*Step 11*). We *hypothesize* that free peptide 15-42 will bind to VE-cadherin between endothelial cells to block endothelial cell binding to 15-42 on intact fibrin(ogen) found in the tumor stroma or tumor vessels, thereby inhibiting tumor-associated angiogenesis (*Step 2*), intravasation (*Step 3*), extravasation (*Step 8*), and angiogenesis at metastatic tumor sites (*Step 10*) (**as denoted by the lightening bolts at these steps in Fig. 10**).

Fig. 10. Schematic summarizing role of fibrin(ogen) 15-42 in breast cancer metastasis and hypothesis development for employing free peptide 15-42 as a therapeutic strategy to treat metastatic breast cancers*.*

Successful demonstration of peptide 15-42 as an inhibitor of breast cancer metastasis and tumor-associated inflammation and angiogenesis *in vivo* would significantly impact breast cancer treatment in a timely manner. Peptide 15-42, an endogenous fragment of fibrin, is already shown to be well tolerated in humans and effective in reducing damage to heart muscle after a heart attack in preclinical models of IR injury. However, until now, no one has proposed the use of peptide 15-42 as an inhibitor of breast cancer metastasis. A precedent and pipeline for production of viable therapeutics based on peptide 15-42 exists for treatment of damaged heart tissue, and Phase I and Phase II clinical trials are ongoing to test the safety and efficacy, respectively, of free 15-42 peptide for IR injury (Hallen et al., 2010; Petzelbauer et al., 2005a; Petzelbauer et al., 2005b; Roesner et al., 2007; Roesner et al., 2009; Wiedemann et al., 2010; Zacharowski et al., 2006). Therefore, the timeline for

The Role of Fibrin(ogen) in Transendothelial Cell Migration During Breast Cancer Metastasis 199

with poor survival (Hattrup & Gendler, 2006; Yuan et al., 2007). MUC1 is upregulated and hypoglycosylated in breast cancers. The polarized expression of MUC1 is lost on cancer cells such that it is expressed on the entire cell surface (Kondo et al., 1998; Moase et al., 2001; Wesseling et al., 1996; Yang et al., 2007). The MUC1 extracellular domain protrudes ~200 nm above the cell surface, whereas most cell surface receptors are ~35 nm long (Wesseling et al., 1996). When MUC1 is interspersed between adhesion molecules, it nonspecifically reduces cell-cell and cell-ECM interactions in vitro and in vivo, likely by steric hindrance caused by the extreme length and high density of the MUC1 at the cell surface (Wesseling et al., 1996) (**Fig. 11B**). MUC1 expression is found on MCF-7, MDA-MB-231, as well as other types of breast cancer cells, particularly on those isolated from patients with a highly aggressive subtype called inflammatory breast cancer (Alpaugh et al., 2002; Schroeder et al., 2003; Walsh et al., 1999); elevated expression of MUC1 contributes to lymphovascular tumor

invasion of inflammatory breast cancer cells (Alpaugh et al., 2002).

cell adhesion in breast epithelial cells.

Fig. 11. Putative mechanisms whereby nonmalignant breast epithelial cells switch to a metastatic breast cancer cell phenotype responsive to fibrinogen-induced TEM. Panel A, schematic depicting loss of function due to release of cell-surface HSPG. Panel B, schematic depicting gain of function by overexpression of MUC1 leading to loss of polarity and cell-

We predict that Fg could bind to normal breast cell surface HSPG through Fg 15-42, thus preventing Fg 15-42 binding to VE-cadherin extracellular domain 3 (EC3) and inhibition of TEM. Enhanced heparanase expression and enzymatic digestion of HSPG in human tumors correlates with metastatic potential, tumor vascularity, and reduced postoperative survival of cancer patients (Vlodavsky et al., 2008). Heparanase-induced loss of breast epithelial cell surface HSPG during conversion of non- or pre-malignant to malignant breast cancers would allow Fg 15-42 binding to VE-cadherin at cell-cell junctions to induce EC permeability. Fg would also bind to breast cancer cell integrins via binding sites on Fg Cterminal domains (**see Fig. 4**) then movement of VE-cadherin (induced by Fg binding to VEcadherin as shown in **Fig. 8B**) in the endothelial cell membrane would induce paracellular transfer of Fg-bound breast cancer cells across the endothelial cell barrier to promote cancer metastasis. A precedent for this mechanism is already established; Fg binding to a counter adhesion molecule facilitates neutrophil TEM 20- to 30-fold (Languino et al., 1995). Overexpression of MUC1 could block accessibility of HSPG on breast cancer cells, which

successful translational to a therapeutic agent to treat metastatic disease in breast cancer patients with different subtypes of the disease would be significantly shortened. Moreover, even if the primary tumor develops its own blood supply before adjuvant therapy with peptide 15-42 is begun, we predict that free peptide 15-42 will prevent subsequent steps required for metastatic spread and growth of breast cancers. Another advantage to this therapeutic strategy is that peptide 15-42 functions outside the cell, precluding the need to deliver the peptide inside cells. Identifying molecular targets for therapeutic intervention of breast cancer metastasis, recruitment of inflammatory cells and angiogenesis will increase long-term disease-free survival and improve the quality of life for breast cancer patients.

#### **4.2 Putative mechanisms whereby nonmalignant breast epithelial cells switch to a metastatic breast cancer cell phenotype responsive to fibrinogen induced TEM**

A class of molecules found in the ECM, inside cells and attached to cell surfaces, called heparan sulfate proteoglycans (HSPG), contribute to breast cancer progression by promoting cancer cell proliferation, TEM, and tumor-associated angiogenesis (Koo et al., 2008). The ability to affect any one of these functions would help to reduce breast cancer metastasis; however, if all three of the functions could be targeted with one therapeutic approach, the morbidity and mortality due to metastatic breast cancer could be significantly reduced. Heparin is widely used as an anticoagulant, but it also inhibits HSPG-dependent mechanisms of cancer metastasis (Levy-Adam et al., 2005). However, anti-metastatic heparins that also inhibit blood coagulation are, therefore, not good candidates for widespread use to treat metastatic breast cancer due to bleeding complications. Thus, another molecular target to inhibit the prometastatic effects of HSPG but not inhibit coagulation is greatly needed. Spontaneous blood-borne and lymphatic metastasis of tumor emboli requires fibrin(ogen) (Palumbo et al., 2002). In addition to binding to VE-cadherin (Yakovlev et al., 2003), Fg 15-42 also binds to heparin and HSPG on endothelial cells with high affinity (Odrljin et al., 1996a; Odrljin et al., 1996b); however, a role for HSPG in Fgmediated breast cancer metastasis has not been studied. Fg binding to heparin and HSPG involves residues 15-42, and 15-42-dependent fibrin binding to EC surfaces can be inhibited with heparin and heparan sulfate but not with chondroitin sulfate, indicating that Fg-15-42 represents a HBD (Odrljin et al., 1996a; Odrljin et al., 1996b). The Fg HBD was later mapped to residues 15-57, which includes the 15-42 VE-cadherin binding domain (Yakovlev et al., 2003; Yakovlev & Medved, 2009). In our recent publication (Sahni et al., 2009), we unexpectedly discovered that Fg enhanced TEM of only malignant breast cancer cells (MCF-7 and MDA-MB-231) but not nonmalignant breast epithelial cells (MCF-10A), suggesting inherent differences in the ability of cancer vs. normal breast epithelial cells to interact with fibrin(ogen). Because TEM of nonmalignant epithelial cells (MCF-10A) could not be enhanced in the presence of Fg (Sahni et al., 2009), we hypothesize that loss of HSPG from the surface of premalignant breast epithelial cells serves as a molecular switch to induce a highly aggressive, metastatic breast cancer phenotype (**Fig. 11A**). We plan to investigate this hypothesis in future studies.

Another mechanism to regulate Fg-enhanced TEM of malignant breast cancer cells is a gain in function of cancer-associated Mucin-1 (MUC1), which is a membrane-associated mucin expressed at low levels on the apical surface of normal polarized epithelial cells. MUC1 is a tumor-associated glycoprotein aberrantly expressed in >90% of breast cancers (Singh & Bandyopadhyay, 2007), promotes cancer cell proliferation and metastasis, and is associated

successful translational to a therapeutic agent to treat metastatic disease in breast cancer patients with different subtypes of the disease would be significantly shortened. Moreover, even if the primary tumor develops its own blood supply before adjuvant therapy with peptide 15-42 is begun, we predict that free peptide 15-42 will prevent subsequent steps required for metastatic spread and growth of breast cancers. Another advantage to this therapeutic strategy is that peptide 15-42 functions outside the cell, precluding the need to deliver the peptide inside cells. Identifying molecular targets for therapeutic intervention of breast cancer metastasis, recruitment of inflammatory cells and angiogenesis will increase long-term disease-free survival and improve the quality of life for breast cancer patients.

**4.2 Putative mechanisms whereby nonmalignant breast epithelial cells switch to a metastatic breast cancer cell phenotype responsive to fibrinogen induced TEM** 

hypothesis in future studies.

A class of molecules found in the ECM, inside cells and attached to cell surfaces, called heparan sulfate proteoglycans (HSPG), contribute to breast cancer progression by promoting cancer cell proliferation, TEM, and tumor-associated angiogenesis (Koo et al., 2008). The ability to affect any one of these functions would help to reduce breast cancer metastasis; however, if all three of the functions could be targeted with one therapeutic approach, the morbidity and mortality due to metastatic breast cancer could be significantly reduced. Heparin is widely used as an anticoagulant, but it also inhibits HSPG-dependent mechanisms of cancer metastasis (Levy-Adam et al., 2005). However, anti-metastatic heparins that also inhibit blood coagulation are, therefore, not good candidates for widespread use to treat metastatic breast cancer due to bleeding complications. Thus, another molecular target to inhibit the prometastatic effects of HSPG but not inhibit coagulation is greatly needed. Spontaneous blood-borne and lymphatic metastasis of tumor emboli requires fibrin(ogen) (Palumbo et al., 2002). In addition to binding to VE-cadherin (Yakovlev et al., 2003), Fg 15-42 also binds to heparin and HSPG on endothelial cells with high affinity (Odrljin et al., 1996a; Odrljin et al., 1996b); however, a role for HSPG in Fgmediated breast cancer metastasis has not been studied. Fg binding to heparin and HSPG involves residues 15-42, and 15-42-dependent fibrin binding to EC surfaces can be inhibited with heparin and heparan sulfate but not with chondroitin sulfate, indicating that Fg-15-42 represents a HBD (Odrljin et al., 1996a; Odrljin et al., 1996b). The Fg HBD was later mapped to residues 15-57, which includes the 15-42 VE-cadherin binding domain (Yakovlev et al., 2003; Yakovlev & Medved, 2009). In our recent publication (Sahni et al., 2009), we unexpectedly discovered that Fg enhanced TEM of only malignant breast cancer cells (MCF-7 and MDA-MB-231) but not nonmalignant breast epithelial cells (MCF-10A), suggesting inherent differences in the ability of cancer vs. normal breast epithelial cells to interact with fibrin(ogen). Because TEM of nonmalignant epithelial cells (MCF-10A) could not be enhanced in the presence of Fg (Sahni et al., 2009), we hypothesize that loss of HSPG from the surface of premalignant breast epithelial cells serves as a molecular switch to induce a highly aggressive, metastatic breast cancer phenotype (**Fig. 11A**). We plan to investigate this

Another mechanism to regulate Fg-enhanced TEM of malignant breast cancer cells is a gain in function of cancer-associated Mucin-1 (MUC1), which is a membrane-associated mucin expressed at low levels on the apical surface of normal polarized epithelial cells. MUC1 is a tumor-associated glycoprotein aberrantly expressed in >90% of breast cancers (Singh & Bandyopadhyay, 2007), promotes cancer cell proliferation and metastasis, and is associated with poor survival (Hattrup & Gendler, 2006; Yuan et al., 2007). MUC1 is upregulated and hypoglycosylated in breast cancers. The polarized expression of MUC1 is lost on cancer cells such that it is expressed on the entire cell surface (Kondo et al., 1998; Moase et al., 2001; Wesseling et al., 1996; Yang et al., 2007). The MUC1 extracellular domain protrudes ~200 nm above the cell surface, whereas most cell surface receptors are ~35 nm long (Wesseling et al., 1996). When MUC1 is interspersed between adhesion molecules, it nonspecifically reduces cell-cell and cell-ECM interactions in vitro and in vivo, likely by steric hindrance caused by the extreme length and high density of the MUC1 at the cell surface (Wesseling et al., 1996) (**Fig. 11B**). MUC1 expression is found on MCF-7, MDA-MB-231, as well as other types of breast cancer cells, particularly on those isolated from patients with a highly aggressive subtype called inflammatory breast cancer (Alpaugh et al., 2002; Schroeder et al., 2003; Walsh et al., 1999); elevated expression of MUC1 contributes to lymphovascular tumor invasion of inflammatory breast cancer cells (Alpaugh et al., 2002).

Fig. 11. Putative mechanisms whereby nonmalignant breast epithelial cells switch to a metastatic breast cancer cell phenotype responsive to fibrinogen-induced TEM. Panel A, schematic depicting loss of function due to release of cell-surface HSPG. Panel B, schematic depicting gain of function by overexpression of MUC1 leading to loss of polarity and cellcell adhesion in breast epithelial cells.

We predict that Fg could bind to normal breast cell surface HSPG through Fg 15-42, thus preventing Fg 15-42 binding to VE-cadherin extracellular domain 3 (EC3) and inhibition of TEM. Enhanced heparanase expression and enzymatic digestion of HSPG in human tumors correlates with metastatic potential, tumor vascularity, and reduced postoperative survival of cancer patients (Vlodavsky et al., 2008). Heparanase-induced loss of breast epithelial cell surface HSPG during conversion of non- or pre-malignant to malignant breast cancers would allow Fg 15-42 binding to VE-cadherin at cell-cell junctions to induce EC permeability. Fg would also bind to breast cancer cell integrins via binding sites on Fg Cterminal domains (**see Fig. 4**) then movement of VE-cadherin (induced by Fg binding to VEcadherin as shown in **Fig. 8B**) in the endothelial cell membrane would induce paracellular transfer of Fg-bound breast cancer cells across the endothelial cell barrier to promote cancer metastasis. A precedent for this mechanism is already established; Fg binding to a counter adhesion molecule facilitates neutrophil TEM 20- to 30-fold (Languino et al., 1995). Overexpression of MUC1 could block accessibility of HSPG on breast cancer cells, which

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