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ARNT-dependent HIF-2 transcriptional activity is not sufficient to regulate downstream target genes in neuroblastoma

Persson C.U., von Stedingk K., Fredlund E., Bexell D., Påhlman S., Wigerup C. Mohlin S.
Experimental cell research, 2020

Hypoxia-inducible factor (HIF)-2α associates with poor outcomes in neuroblastoma and glioblastoma, and gain-of-function mutations in the EPAS1gene (encoding HIF-2α) have been reported in paragangliomas and pheochromocytomas. Specific targeting of a druggable hydrophobic pocket in the HIF-2αPAS-B domain with PT2385 has demonstrated promising clinical results for clear cell renal cell carcinoma (ccRCC). Here, the authors investigated the effect of PT2385-mediated inhibition of ARNT dependent HIF-2 activity. Neuroblastoma patient-derived xenograft (PDX) cells cultured on Biolaminin 521 were treated with PT2385 and analyzed for HIF-2-dependent gene expression, HIF activity, HIF-2αprotein localization, response to chemotherapy and orthotopic tumor growth in vivo. The authors detected high levels of HIF-2α protein in perivascular niches in neuroblastoma PDXs in vivo and at oxygenated conditions in PDX-derived cell cultures in vitro, particularly in the cytoplasmic fraction. Nuclear HIF-2αexpression was reduced following PT2385 treatment, but surprisingly, virtually no effects on tumor growth in vivo or expression of canonical HIF downstream target genes in vitro were observed. RNA sequencing of PT2385-treated PDX cells revealed a virtually unaffected transcriptome. Treatment with PT2385 did not affect cellular response to chemotherapy. In contrast, HIF-2αprotein knockdown resulted in profound downregulation of target genes. The lack of effect from PT2385 treatment in combination with high cytoplasmic HIF-2αexpressionat normoxia suggests that HIF-2α has additional roles than acting as an ARNT dependent transcription factor. It is important to further unravel the conditions at which HIF-2αhas transcriptional and non-transcriptional roles in neuroblastoma.

 

Collaboration of 3D Context and Extracellular Matrix in the Development of Glioma Stemness in a 3D Model

Ma N.K.L., Kai Lim J., Fatt Leong M., Sandanaraj E., Ti Ang B., Tang C., Wan A.C.A.
Biomaterials, 2015

The results demonstrate how 3D versus 2D context profoundly affects ECM signaling, leading to stemness. U251 glioblastoma cells were cultured on electrospun polystyrene (ESPS) scaffolds coated with 7 different laminin isoforms (LAMscreen kit) to provide a 3D model for stem cell-related genes and proteins expression studies. The authors observed collaboration between 3D context and laminins in promoting glioma stemness. The results indicate the influence of 3D (versus 2D) context on stemness markers and integrin expression, specifically, the upregulation of the laminin-binding integrins a6b4. Enhanced clonogenicity of cells grown on ESPS scaffolds in collaboration with laminins -411, -421, -511 and -521.

 

A laminin 511 matrix is regulated by TAZ and functions as the ligand for the a6Bb1 integrin to sustain breast cancer stem cells

Chang C., Lal Goel H., Gao H., Pursell B., Shultz L.D., Greiner D.L., Ingerpuu S., Patarroyo M., Cao S., Lim E., Mao J., Kulju McKee K., Yurchenco P.D., Mercurio A.M.
Research communication, 2015

One of the first papers that highlighted the importance of ECM proteins in 2D breast cancer stem cell culture. Shows that laminin-511 is an acritical niche component for breast cancer stem cells. Breast cancer stem cells produce a laminin-511 matrix that functions as the ligand for the a6Bb1 integrin to promote self-renewal and tumor initiation. The authors observed that TAZ regulates the transcription of the a5 subunit of laminin-511 and the formation of a laminin-511 matrix. These data establish a positive feedback loop involving TAZ and laminin-511 that contributes to stemness in breast cancer. They see down-regulation of the laminin B2 chain.

 

Laminin-421 produced by lymphatic endothelial cells induces chemotaxis for human melanoma cells

Saito N., Hamada J., Furukawa H., Tsutsumida A., Oyama A., Funayama E., Saito A., Tsuji T., Tada M., Moriuchi T., Yamamoto Y.
Pigment Cell Melanoma Res., 2009

Here, the authors investigate the molecular mechanism of lymphatic metastasis. They examined the influence of interactions between normal lymphatic endothelial cells (LECs) and melanoma cells on cell migration. LEC conditioned medium (LEC-CM) contained chemotactic and chemokinetic activities for human melanoma cell lines. The chemotactic activity of LEC-CM was abolished by immunodepletion with anti-laminin-1 antibody. And immunoprecipitation and Western blot analyses revealed that LEC-CM contained laminin a4 and 5, b1 and 2, and c1, corresponding to isoforms -521, -511, -421 and -411. When melanoma C8161 cells were treated with function-blocking antibodies to integrin a3 or a6, their chemotactic responses to LEC-CM were markedly reduced. Furthermore, the knock-down of tetraspanin CD151 weakened the chemotactic responses of C8161 and MeWo cells to LEC-CM. These data suggest that laminin secreted by LEC possibly facilitates lymphatic metastasis through the induction of chemotaxis of melanoma cells.

 

L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer

Ganesh K., Basnet H., Kaygusuz Y., Laughney A.M., He L., Sharma R., O’Rourke K.P., Reuter V.P., Huang Y.-H., Turkekul M., Er E.E., Masilionis I., Manova-Todorova K., Weiser M.R., Saltz L.B., Garcia-Aguilar J., Koche R., Lowe S.W., Pe’er D., Shia J., Massagué J.
Nature Cancer, 2020

The authors show that L1CAM+ cells in human colorectal cancer (CRC) have metastasis-initiating capacity, and they define their relationship to tissue regeneration. By using recombinant L1CAM extracellular domain and basement membrane components, they confirmed that L1CAM bound heterophilically to laminins known to be expressed in the intestinal and endothelial cell basement membranes in addition to exhibiting homophilic interaction with L1CAM itself. L1CAM knockdown inhibited the ability of CRC organoid-derived cells to bind to laminin-coated plates. Together, these data suggest that L1CAM enables the adhesion of metastasis-initiating cells to laminin-rich basement membranes, which is required for metastasis and organoid growth.

 

Laminin 521 enhances self-renewal via STAT3 activation and promotes tumor progression in colorectal cancer

Qin Y, Shembrey C, Smith J, Paquet-Fifield S, Behrenbruch C, Beyit LM, Thomson BNJ, Heriot AG, Cao Y, Hollande F.
Cancer Lett. 2020

Remodeling of basement membrane proteins contributes to tumor progression towards the metastatic stage. Here, the authors show that one of these proteins, laminin 521 (LN521), promotes colorectal cancer (CRC) cell self-renewal and invasion. siRNA-mediated knockdown of endogenously-produced laminin alpha 5, as well as treatment with neutralizing antibodies against integrin α3β1 and α6β1, were able to reverse the effect of LN521 on self-renewal. Exposure of CRC cells to LN521 enhanced STAT3 phosphorylation, and incubation with STAT3 inhibitors Napabucasin and Stattic were sufficient to block the LN521-driven self-renewal increase. Robust expression of laminin alpha 5 was detected in 7/10 liver metastases tissue sections collected from CRC patients as well as in mouse liver metastasis xenografts, in most cases within areas expressing metastasis cancer stem cell markers such as c-KIT and CD44v6. Finally, retrospective analysis of multiple CRC datasets highlighted the significant association between high LN521 mRNA expression and poor clinical outcome in colorectal cancer patients. Collectively our results indicate that high Laminin 521 expression is a frequent feature of metastatic dissemination in CRC and that it promotes cell invasion and self-renewal, the latter through the engagement of integrin isoforms and activation of STAT3 signaling.

 

Laminins and cancer stem cells: Partners in crime?

Qin Y., Rodin S., Simonson O.E., Hollande F.
Seminars in Cancer Biology, 2016

A review that discusses the role of laminin as a regulator of cancer stem cells, in tumor progression and drug resistance. A growing body of literature suggests that laminins may act as regulators of cancer stem cells, a tumor cell subpopulation that plays an instrumental role in long-term cancer maintenance, metastasis development, and therapeutic resistance. The accumulating evidence in this emerging research area suggests that laminins represent potential therapeutic targets for anti-cancer treatments against cancer stem cells and that they may be used as predictive and prognostic markers to inform clinical management and improve patient survival.

 

Gelatine methacrylamide-based hydrogels – an alternative 3D cancer cell culture system

Kaemmerer E., Melchels F.P.W, Holzapfel B.M, Meckel T., Hutmacher D.W., Loessner D.
Acta Biomaterialia, 2014

The authors present a 3D biomaterial platform for the analysis of ovarian cancer spheroid growth that is an efficient semi-synthetic alternative, combining native ECM components and tunable matrix properties, resulting in higher reproducibility, less complexity and better comparability between different groups than traditional cell monolayer approaches. In this study, gelatine methacrylamide-based hydrogels (GelMA) with added LN-411 were established as in vitro and in vivo spheroid-based 3D cancer models.

 

Laminin Interactions with Head and Neck Cancer Cells under Low Fluid Shear Conditions Lead to Integrin Activation and Binding

Fennewald S.M., Kantara C., Sastry S.K., Resto V.A
Journal of biological chemistry, 2012

Lymphatic metastasis of cancer cells involves movement from the primary tumor site to the lymph node, where the cells must be able to productively lodge and grow. Head and neck squamous cell carcinoma (HNSCC) cell lines cultured on placental laminin (laminin-511 is the major laminin), laminin-332 purified from human foreskin keratinocytes and human recombinant laminin-511, -211, -111, and -411. HNSCC cell lines bound to laminin-511 and -211 but also to -411 to a lower extent, under lymphodynamic low shear stress (0.07 dynes/cm2), consistent with lymph flow. Binding only occurred in the presence of shear stress and not in the absence of flow. The authors conclude that B1 integrins mediate tumor cell/lymph node interactions active under lymphodynamic flow. These interactions may drive growth and immunomodulation in this niche.

 

Ultraviolet-radiation-induced inflammation promotes angiotropism and metastasis in melanoma

Bald T., Quast T., Landsberg J., Rogava M., Glodde n., Lopez-Ramos D., Kohlmeyer J., Riesenberg S., van den Boorn-Konijnenberg D.,  Hömig-Hölzel C., Reuten R., Schadow B., Weighardt H., Wenzel D., Helfrich I., Schadendorf D., Bloch W., Bianchi M.E., Lugassy C., Barnhill R.L., Koch M., Fleischmann B.K., Förster I., Kastenmüller W., Kolanus W., Hölzel M., Gaffal E., Tüting T.
Nature Letter. 2014

Here we report that repetitive UV exposure of primary cutaneous melanomas in a genetically engineered mouse model. UV irradiation enhanced the expansion of tumor cells along abluminal blood vessel surfaces and increased the number of lung metastases, depended on the recruitment and activation of neutrophils. In a static cell adhesion assays, cells were allowed to adhere to the various matrices: fibronectin-1, collagen type I, collagen type IV, laminin-111 (Sigma), laminin-411 or laminin-511.  An inflammatory environment promotes the ability of mouse and human melanoma cells to migrate towards endothelial cells and use selective motility cues on their surfaces.

The UV-induced neutrophilic inflammatory response stimulated angiogenesis and promoted the ability of melanoma cells to migrate towards endothelial cells and use selective motility cues on their surfaces. Our results not only reveal how UV irradiation of epidermal keratinocytes is sensed by the innate immune system but also show that the resulting inflammatory response catalyzes reciprocal melanoma–endothelial cell interactions leading to perivascular invasion, a phenomenon originally described as angiotropism in human melanomas by histopathologists.

 

Polymerized Laminin-332 Matrix Supports Rapid and Tight Adhesion of Keratinocytes, Suppressing Cell Migration

Kariya Y., Sato H., Katou N., Kariya Y., Miyazaki K.
PLOS ONE, 2012

Laminin-332 is known to supports the stable anchoring of basal keratinocytes to the epidermal basement membrane but is also a motility factor for wound healing and cancer invasion. Here they investigated Laminin-332 matrices deposited by normal human keratinocytes and many cancer cell lines. All types of cells efficiently deposited Laminin-332 on the culture plates in specific patterns. On the contrary, laminins containing laminin ß1 and/or c1 chains (such as laminin-511 and laminin-311) were not deposited on the culture plates even if secreted into the culture medium. The deposited laminin-332 matrix showed a mesh-like network structure as analyzed by electron microscopy, suggesting that laminin-332 was highly polymerized. Laminin-332 matrix rather suppressed the migration of keratinocytes as compared with purified laminin-332, which highly promoted cell migration. The Lm332 matrix supports the adhesion of keratinocytes much more strongly and stably than purified laminin-332. Integrin a3ß1 bound to the laminin-332 matrix at a three times higher level than purified laminin-332. These results indicate that the polymerized laminin-332 matrix supports stable cell adhesion whereas unassembled soluble laminin-332 supports cell migration. The question is though how the purified laminin-332 looked like. Difficult to purify and might be fractionated.

 

Laminin-332 sustains chemoresistance and quiescence as part of the human hepatic cancer stem cell niche

Govaere O., Wouters J., Petz M., Vandewynckel Y-P., Van den Eynde K., Van den broeck A., Verhulst S., Dollé L., Gremeaux L., Ceulemans A., Nevens F., van Grunsven L.A., Topal B., Vankelecom H., Giannelli G., Van Vlierberghe H. Mikulits W., Komuta M., Roskams T.
Journal of hepatology, 2015

This study demonstrates that tumor behavior is plastic and depends on the microenvironment of the tumor cell. We particularly identified an important role for laminin-332 and more specifically its gamma2-chain as part of the specialized cancer stem cell niche in maintaining and supporting ‘stemness’, e.g. quiescence and chemo-resistance. laminin-332 induces K19 expression, quiescence, and chemo-resistance in vitro. Laminin-332 not only protects hepatic cancer cells against chemotherapy but stimulates cell proliferation upon sorafenib exposure. Therefore, monoclonal antibody treatment targeting the gamma2-chain of laminin-332 could provide an innovative therapy of hepatic cancer.

 

Monoclonal antibodies to human laminin α4 chain globular domain inhibit tumor cell adhesion and migration on laminins 411 and 421, and binding of α6β1 integrin and MCAM to α4-laminins

Ishikawa T., Wondimu Z., Oikawa Y., Ingerpuu S., Virtanen I., Patarroyo M.
Matrix Biology, 2014

α4-Laminins, such as laminins -411 and -421, are mesenchymal laminins expressed by vascular and lymphatic endothelial cells, leukocytes and other normal cell types. These laminins are recognized by α6β1 and α6β4 integrins and MCAM (CD146) and promote adhesion and migration of the cells. α4-Laminins are also expressed and secreted by some tumor cells and strongly promote tumor cell migration. Moreover, the abluminal side of blood and/or lymphatic vessels and the nerve perineurium, common tracks of tumor cell dissemination, express α4-laminins, and these laminin isoforms, when expressed in the stroma, may contribute to tumor invasion. In the present study, we examined ten mAbs to human laminin α4 chain for their reactivity with the isolated laminin α4 globular domain, their ability to inhibit tumor cell adhesion and migration on laminin-411 and -421, and their effect on the binding of α6β1 integrin and MCAM to both α4-laminins. The results indicate that mAbs to the laminin α4 globular domain are able to inhibit tumor cell adhesion and migration on laminin-411 and -421 and that α6β1 integrin and MCAM bind α4-laminins at very close sites on the globular domain. These reagents contribute to a better understanding of the biology of α4-laminins and may have a therapeutic potential in malignant and inflammatory diseases.

 

Selection and Characterization of an α6β4 Integrin blocking DNA Aptamer

Berg K., Lange T., Mittelberger F., Schumacher U., Hahn U.
Molecular Therapy—Nucleic Acids, 2016

Cancer cells use the α6β4 integrin/laminin-332 interaction to activate signaling pathways promoting tumor cell growth, invasion and metastasis, the inhibition of this interaction is of high therapeutic interest. Here, the authors report on the selection of a DNA aptamer inhibiting the interaction between α6β4 integrin and laminin-332. This Integrin α6β4-specific DNA Aptamer inhibits the adhesion of prostate cancer cells (PC-3) to laminin-332 with an IC50 value of 149 nmol/l. the aptamer was internalized into PC- 3-cells. Further characterization showed specificity to α6 integrins and a half-life in the murine blood plasma of 6 hours.

 

Ligand-binding specificities of laminin-binding integrins: a comprehensive survey of laminin-integrin interactions using recombinant alpha3beta1, alpha6beta1, alpha7beta1 and alpha6beta4 integrins

Nishiuchi et al.
Matrix Biol., 2006 

 

The opposing roles of laminin-binding integrins in cancer

Ramovs V., te Molder L., Sonnenberg A.
Matrix Biology, 2016

In this review the authors discuss the dual role of the laminin-binding integrins α3β1 and α6β4 in tumor development and progression and examine the factors and mechanisms involved in these opposing effects. 

 

Effect of laminin-332 on motility and invasion in bladder cancer

Kang et al.
Kaohsiung J. Med. Sci., 2013


Assessment of laminin-5 in oral dysplasia and squamous cell carcinoma

Rani et al.
J. Oral Maxillofac., 2013



Molecular organization of the cutaneous basement membrane zone

Ghohestani et al. 
Clin Dermatol., 2001


Current insights into the formation and breakdown of hemidesmosomes

Litjens et al.
Trends Cell Biol., 2006