Evasive mechanisms triggered by the tyrosine kinase inhibitor sorafenib reduce its efficacy in hepatocellular carcinoma (HCC) treatment. Drug-resistant cancer cells frequently exhibit sphingolipid dysregulation, reducing chemotherapeutic cytotoxicity via the induction of ceramide-degrading enzymes. However, the role of ceramide in sorafenib therapy and resistance in HCC has not been clearly established. Our data reveals that ceramide-modifying enzymes, particularly glucosylceramide synthase (GCS), are upregulated during sorafenib treatment in hepatoma cells (HepG2 and Hep3B), and more importantly, in sorafenib-resistant cell lines. GCS silencing or pharmacological GCS inhibition sensitized hepatoma cells to sorafenib exposure. GCS inhibition, combined with sorafenib, triggered cytochrome c release and ATP depletion in sorafenib-treated hepatoma cells, leading to mitochondrial cell death after energetic collapse. Conversely, genetic GCS overexpression increased sorafenib resistance. Of interest, GCS inhibition improved sorafenib effectiveness in a xenograft mouse model, recovering drug sensitivity of sorafenib-resistant tumors in mice. In conclusion, our results reveal GCS induction as a mechanism of sorafenib resistance, suggesting that GCS targeting may be a novel strategy to increase sorafenib efficacy in HCC management, and point to target the mitochondria as the subcellular location where sorafenib therapy could be potentiated.

Hepatocellular carcinoma (HCC) frequently develops in a pro-inflammatory and pro-fibrogenic environment with hepatic stellate cells (HSCs) remodeling the extracellular matrix composition. Molecules secreted by liver tumors contributing to HSC activation and peritumoral stromal transformation remain to be fully identified. Here we show that conditioned medium from HCC cell lines, Hep3B and HepG2, induced primary mouse HSCs transdifferentiation, characterized by profibrotic properties and collagen modification, with similar results seen in the human HSC cell line LX2. Moreover, tumor growth was enhanced by coinjection of HepG2/LX2 cells in a xenograft murine model, supporting a HCC-HSC crosstalk in liver tumor progression. Protein microarray secretome analyses revealed angiogenin as the most robust and selective protein released by HCC compared to LX2 secreted molecules. In fact, recombinant angiogenin induced in vitro HSC activation requiring its nuclear translocation and rRNA transcriptional stimulation. Moreover, angiogenin antagonism by blocking antibodies or angiogenin inhibitor neomycin decreased in vitro HSC activation by conditioned media or recombinant angiogenin. Finally, neomycin administration reduced tumor growth of HepG2-LX2 cells coinjected in mice. In conclusion, angiogenin secretion by HCCs favors tumor development by inducing HSC activation and ECM remodeling. These findings indicate that targeting angiogenin signaling may be of potential relevance in HCC management.

Basement membrane (BM) zone-associated collagen XV (ColXV) has been shown to suppress the malignancy of tumour cells, and its restin domain can inhibit angiogenesis. In human breast cancer, as well as in many other human carcinomas, ColXV is lost from the epithelial BM zone prior to tumour invasion. Here, we addressed the roles of ColXV in breast carcinogenesis using the transgenic MMTV-PyMT mouse mammary carcinoma model. We show here for the first time that the inactivation of Col15a1 in mice leads to changes in the fibrillar tumour matrix and to increased mammary tumour growth. ColXV is expressed by myoepithelial and endothelial cells in mammary tumours and is lost from the ductal BM along with the loss of the myoepithelial layer during cancer progression while persisting in blood vessels and capillaries, even in invasive tumours. However, despite the absence of anti-angiogenic restin domain, neovascularisation was reduced rather than increased in the ColXV-deficient mammary tumours compared to controls. We also show that, in robust tumour cell transplantation models or in a chemical-induced fibrosarcoma model, the inactivation of Col15a1 does not affect tumour growth or angiogenesis. In conclusion, our results support the proposed tumour suppressor function of ColXV in mammary carcinogenesis and reveal diverse roles of this collagen in different cancer types.

Integrin α11β1 is a collagen-binding integrin that is needed to induce and maintain the myofibroblast phenotype in fibrotic tissues and during wound healing. The expression of the α11 is upregulated in cancer-associated fibroblasts (CAFs) in various human neoplasms. We investigated α11 expression in human cutaneous squamous cell carcinoma (cSCC) and in benign and premalignant human skin lesions and monitored its effects on cSCC development by subjecting α11-knockout (Itga11−/−) mice to the DMBA/TPA skin carcinogenesis protocol. α11-deficient mice showed significantly decreased tumor cell proliferation, leading to delayed tumor development and reduced tumor burden. Integrin α11 expression was significantly upregulated in the desmoplastic tumor stroma of human and mouse cSCCs, and the highest α11 expression was detected in high-grade tumors. Our results point to a reduced ability of α11-deficient stromal cells to differentiate into matrix-producing and tumor-promoting CAFs and suggest that this is one causative mechanism underlying the observed decreased tumor growth. An unexpected finding in our study was that, despite reduced CAF activation, the α11-deficient skin tumors were characterized by the presence of thick and regularly aligned collagen bundles. This finding was attributed to a higher expression of TGFβ1 and collagen crosslinking lysyl oxidases in the Itga11-/- tumor stroma. In summary, our data suggest that α11β1 operates in a complex interactive tumor environment to regulate ECM synthesis and collagen organization and thus foster cSCC growth. Further studies with advanced experimental models are still needed to define the exact roles and molecular mechanisms of stromal α11β1 in skin tumorigenesis.

The tumor extracellular matrix (ECM) is a critical regulator of cancer progression and metastasis, significantly affecting the treatment response. Expression of collagen XVIII (ColXVIII), a ubiquitous component of basement membranes, is induced in many solid tumors, but its involvement in tumorigenesis has remained elusive. We show here that ColXVIII is markedly upregulated in human breast cancer (BC) cells and is closely associated with a poor prognosis in high-grade BC, especially in human epidermal growth factor receptor 2 (HER2)-positive and basal/triple-negative cases. We identified a novel mechanism of action for ColXVIII as a modulator of epidermal growth factor receptor (EGFR/ErbB) signaling and show that it forms a complex with EGFR, HER2 and alpha6 integrin to promote cancer cell proliferation in a pathway involving its N-terminal portion and the MAPK/ERK1/2 and PI3K/Akt cascades. In vivo studies with Col18a1 mouse models crossed with the MMTV-PyMT mammary carcinogenesis model showed that the short ColXVIII isoform promotes BC growth and metastasis in a tumor cell-autonomous manner. Moreover, the number of mammary cancer stem cells was significantly reduced in both mouse and human cell models upon ColXVIII inhibition. Finally, ablation of ColXVIII in human BC cells and the MMTV-PyMT model substantially improved the efficacy of certain EGFR/ERbB-targeting therapies, even abolishing resistance to EGFR/ErbB inhibitors in some cell lines. In summary, a new function is revealed for ColXVIII in sustaining the stemness properties of BC cells, and tumor progression and metastasis through EGFR/ErbB signaling, suggesting that targeting ColXVIII in the tumor milieu may have significant therapeutic potential.

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