Mutated HRAS Activates YAP1-AXL Signaling to Drive Metastasis of Head and Neck Cancer

Abstract

The survival rate for patients with head and neck cancer (HNC) diagnosed with cervical lymph node (cLN) or distant metastasis is low. Genomic alterations in the HRAS oncogene are associated with advanced tumor stage and metastasis in HNC. Elucidation of the molecular mechanisms by which mutated HRAS (HRASmut) facilitates HNC metastasis could lead to improved treatment options for patients. Here, we examined metastasis driven by mutant HRAS in vitro and in vivo using HRASmut human HNC cell lines, patient-derived xenografts, and a novel HRASmut syngeneic model. Genetic and pharmacological manipulations indicated that HRASmut was sufficient to drive invasion in vitro and metastasis in vivo. Targeted proteomic analysis showed that HRASmut promoted AXL expression via suppressing the Hippo pathway and stabilizing YAP1 activity. Pharmacological blockade of HRAS signaling with the farnesyltransferase inhibitor tipifarnib activated the Hippo pathway and reduced the nuclear export of YAP1, thus suppressing YAP1-mediated AXL expression and metastasis. AXL was required for HRASmut cells to migrate and invade in vitro and to form regional cLN and lung metastases in vivo. In addition, AXL-depleted HRASmut tumors displayed reduced lymphatic and vascular angiogenesis in the primary tumor. Tipifarnib treatment also regulated AXL expression and attenuated VEGFA and VEGFC expression, thus regulating tumor-induced vascular formation and metastasis. Our results indicate that YAP1 and AXL are crucial factors for HRASmut-induced metastasis and that tipifarnib treatment can limit the metastasis of HNC tumors with HRAS mutations by enhancing YAP1 cytoplasmic sequestration and downregulating AXL expression.

Significance: Mutant HRAS drives metastasis of head and neck cancer by switching off the Hippo pathway to activate the YAP1-AXL axis and to stimulate lymphovascular angiogenesis.

Figure 1:. HRAS^mut regulates invasive potential in vitro via the modulation of AXL through YAP1 activation by switching off Hippo pathway.

A, Western blot confirming the ectopic expression of mutated HRAS^V12 in the non-metastatic Cre-expressing CAL33 cell line. B, Bar graph showing enhanced migration and invasion ability of CAL33 Cre⁺ HRAS^V12+ cells in vitro. C, Western blot showing silencing of HRAS expression in an HRAS^mut HNC-HN31 cell line. D, Silencing of HRAS expression reduces migration and invasive potential of HN31 cells in vitro. E, Volcanic plot showing the effect of altered expression of oncogenic proteins on HRAS gain of function (CAL33) and loss of function (HN31). Proteins with a fold change of more than 1 or less than −1 with a P-value less than 0.001 are shown in the plot. F, Venn diagram showing proteins altered during gain-of-function and loss-of-function manipulations of HRAS in CAL33 and HN31 cell lines. G, Western blot showing effect of enhanced AXL and YAP1 expression on ectopic expression of HRAS^V12 in CAL33 Cre expressing cells. H, Western blot showing effect of AXL and YAP1 downregulation on silencing HRAS expression in HN31 cells. β-actin was used as a loading control. I, Western blot confirming YAP1 silencing in HN31 cells with a concomitant reduction in AXL expression. J, Bar graph showing that YAP1 silencing also reduces the migration and invasion potential of HN31 cells. K, Western blot showing the effect of the expression of various phosphorylated and total proteins in the Hippo pathway, namely, MST1/2, LATS1, YAP1 and TAZ, on silencing HRAS in HN31 cells. L, Western blot showing the downregulation of AXL and YAP1 in response to tipifarnib (1 μM) treatment. M, Tipifarnib treatment of HN31 cells for 24 h reduces the invasive capability of the cells. N, Western blot showing the expression of various phosphorylated and total proteins in the Hippo pathway, namely, MST1, LATS1, YAP1, and TAZ, upon treating HN31 cells with 1 μM tipifarnib for 12 h. O, Nuclear and cytoplasmic expression of YAP1 in response to tipifarnib treatment. Actin and histone H3 were used as cytoplasmic and nuclear protein loading controls, respectively. In the different panels of this figure, statistically significant differences (P value = 0.0001) are indicated by ****. Quantification of western blots are provided as supplementary figure 1B, 1C, and 1F–L.

Figure 2:. HRAS-YAP1-AXL axis determines the metastatic potential of human HNC cells in vivo.

A,B, In vivo analysis of (A) tumor growth kinetics and (B) number of enlarged lymph nodes and lung metastasis foci in mice (n = 12) bearing CAL33 Cre⁺ or CAL33 Cre⁺ HRAS^V12+ tumors; the figures show the enhanced metastasis potential on the incorporation of HRAS mutation. C, Representative immunohistochemistry images of the lung metastases (stained for pan keratin) of CAL33 Cre⁺ and CAL33 Cre⁺ HRAS^V12+. Inset magnification 20×. D, E In vivo analysis of (D) tumor growth kinetics and (E) number of enlarged lymph nodes and lung metastasis foci in mice (n = 12) injected with HRAS^mut HNC cell lines - UMSCC17B or HN31; the figures show the high metastasis potential in HN31 cells. F, Western blot showing the expression of AXL and YAP1 in UMSCC17B and HN31 cell lines. β-actin was used as a loading control. G, H, In vivo representation of (G) tumor growth kinetics, and (H) number of enlarged lymph nodes, and lung metastasis foci in mice (n =12) bearing HRAS mutant HNC PDXs (PDX1 and PDX2) after injection with single cell suspensions into the lip of the NSG mice. I, Western blot showing the expression of AXL and YAP1, in PDXs. J, K, In vivo analysis of (J) relative tumor growth, and (K) the numbers of enlarged lymph nodes and lung metastasis foci in HN31 tumor-bearing mice (n=5–6) treated with tipifarnib (60 mg/kg/twice daily); the findings show a reduced tumor and metastasis burden. L, Representative images of lung macro-metastasis; lungs were isolated from vehicle-and tipifarnib-treated HN31 tumor-bearing mice (n=5). In the different panels of this figure, statistically significant differences are indicated by *, **, ***, or **** for P values of 0.05, 0.01, 0.001 or 0.0001, respectively. Quantification of western blots are provided as supplementary figure 2C and 2E.

Figure 3:. Genetic/pharmacological dysregulation of AXL signaling modulates the metastatic potential of human HRAS^mut HNC cells.

A, Western blot and qPCR confirming the AXL knockdown of HN31 cells. B, Reduced migration capability of HN31 cells. C, Reduced migration capability of HN31 cells upon treatment with anti-Gas6 D, E, In vivo analysis of (D) tumor growth kinetics and (E) number of enlarged lymph nodes and lung metastasis foci in AXL knockdown HN31 tumor-bearing mice (n = 5). F, Bar graphs showing that pharmacological inhibition of AXL signaling by R428 (2 μM) reduces the invasive potential of HN31 cells in vitro. G, H, In vivo analysis of (G) tumor growth kinetics and (H) number of enlarged lymph nodes and lung metastasis foci in HN31 tumor-bearing mice (n = 5) treated with R428 (50 mg/kg/twice daily). I,J, In vivo analysis of (I) tumor growth kinetics and (J) number of enlarged lymph nodes and lung metastasis foci (n = 5) in PDX2 tumor-bearing mice treated with R428 (50 mg/kg/twice daily). K, AXL overexpression (AXL OE) in weakly metastatic UMSCC17B cell line enhances invasive potential in vitro compared to vector control. L, M, In vivo analysis of (L) tumor growth kinetics and (M) number of enlarged lymph nodes and lung metastasis foci in AXL overexpressing UMSCC17B cells injected mice (n=5). In the different panels of this figure, statistically significant differences are indicated by *, **, ***, or **** for P values of 0.05, 0.01, 0.001 or 0.0001, respectively. ns denotes not significant. Quantification of western blots is provided as supplementary figure 3A.

Figure 4:. AXL knockdown in HRAS^mut HNC attenuates vascular/lymphatic angiogenesis by downregulating VEGFA and VEGFC expression.

A, Immunostaining with CD31 and LYVE-1 (markers for angiogenesis and lymphangiogenesis, respectively) in primary tumors of AXL knockdown HN31; the micrographs show minimal angiogenesis and lymphangiogenesis upon AXL downregulation. The percentage of positive cells per field for each stain is provided on the right. B, Representative microscopic images of HUVEC tube formation with conditioned media from AXL knockdown HN31. Bar graphs representing the quantification of total mesh area on tube formation under each condition are shown on the right. C, Fold change in mRNA expression of VEGFA and VEGFC upon AXL knockdown in HN31 cells. D, Representative microscopic images of HUVEC tube formation using conditioned media from HN31 cells treated with R428 (2 μM) or DMSO for 24 h. E, Fold change in mRNA expression of VEGFA and VEGFC upon treatment of HN31 cells with R428 (2 μM). F, G, Representative microscopic images of HUVEC tube formation (F) using conditioned media from shCT HN31 cells treated with Avastin (10μg/ml) and (G) in conditioned media from shAXL HN31 cells treated with VEGFA (20ng/ml). Quantification of total mesh area on tube formation under each condition are shown on the right. H, Fold change in mRNA expression of VEGFA and VEGFC upon treatment of HN31 cells with tipifarnib (1 μM). I, Representative microscopic images of HUVEC tube formation using conditioned media from HN31 cells treated with tipifarnib (1 μM) or DMSO for 24 h. In the different panels of this figure, statistically significant differences ** or **** for P values of 0.01 or 0.0001. ns denotes not significant.

Figure 5:. Metastatic potential of murine HRAS^mut HNC is modulated by AXL.

A, Top: Schematic representation of the generation of HRAS mutant murine cells from primary KRT14 Cre⁺ tongue epithelial cells. Bottom: Western blot showing the expression of E-cadherin, N-cadherin, AXL, YAP1, p53, HRAS^V12, and β-actin in primary tongue epithelial cells (1⁰ EpT) and HRAS^V12 lentiviral transduced tongue epithelial cells (HRAS^V12 shp53 EpT). B, C, In vivo analysis of (B) tumor growth kinetics and (C) number of enlarged lymph nodes and lung metastasis foci of HRAS^V12 shp53 EpT in WT and NSG mice (n=6). D, E, In vivo analysis of tumor growth kinetics, number of enlarged lymph nodes and lung metastasis foci in (D) AXL knockout HRAS^V12 shp53 EpT tumor-bearing WT mice (n = 6) and (E) AXL knockout HRAS^V12 shp53 EpT tumor-bearing NSG mice (n = 6) showing a reduced metastatic burden. F, Immunostaining with CD31 and LYVE-1 in primary tumors of AXL knockout HRAS^V12 shp53 EpT showing minimal angiogenesis and lymphangiogenesis upon AXL downregulation. The percentage of positive cells per field for each staining is shown on the right. G, Representative microscopic images of HUVEC tube formation with conditioned media from AXL knockout HRAS^V12 shp53 EpT. Bar graphs representing the quantification of total mesh area on tube formation under each condition are shown on the right. H, Fold change in mRNA expression of VEGFA and VEGFC upon AXL knockout in HRAS^V12 shp53 EpT cells. In the different panels of this figure, statistically significant differences are indicated by **, ***, or **** for P values of 0.01, 0.001 or 0.0001, respectively. ns denotes not significant. Quantification of western blots are provided as supplementary figure 5D.

Figure 6:. Inhibition of HRAS/AXL signaling diminishes the metastatic potential of murine HRAS^mut cells.

A, Pharmacological inhibition of AXL signaling with R428 reduced the migration and invasion of HRAS^V12 shp53 EpT. B, C, In vivo analysis of tumor growth kinetics, and numbers of enlarged lymph nodes and lung metastasis foci in HRAS^V12 shp53 EpT tumor upon treatment with R428 (50 mg/kg/twice daily) in (B) WT mice (n=5) and (C) NSG mice (n=5), showing R428 treatment reduced the metastatic burden without affecting the tumor growth in both WT and NSG mice. D, Pharmacological inhibition of HRAS signaling with tipifarnib reduced the migration and invasion of HRAS^V12 shp53 EpT. E, F, In vivo analysis of tumor growth kinetics, and numbers of enlarged lymph nodes and lung metastasis foci in HRAS^V12 shp53 EpT tumor-baering WT (E) and NSG (F) mice (n = 6) treated with tipifarnib (60 mg/kg/twice daily) showing tumor growth delay and reduced metastatic burden in both WT and NSG mice. In the different panels of this figure, statistically significant differences are indicated by **, ***, or **** for P values of 0.01, 0.001 or 0.0001, respectively. ns denotes not significant.

Figure 7:. Schematic representation of the molecular mechanism of HRAS-mutation-mediated metastasis in HNC.

Top - In the disease state, HRAS^mut inhibits the Hippo pathway, preventing YAP1 degradation and, in turn, leading to nuclear export of YAP1, thereby regulating the transcriptional expression of multiple genes, including AXL. AXL overexpression and activation by its ligand Gas6 enhance the migratory activity of tumor cells and upregulate the expression of VEGFA, VEGFC and other angiogenic factors as well as EMT genes. This activation of HRAS-YAP1-AXL enhanced angiogenesis, sustained proliferation, and increased metastasis potential, causing metastatic spread to the cLNs and lungs. Middle - Treament of tumors with R428 did not reduce tumor cell proliferation but did reduce the metastasis potential of tumor cells and tumor angiogenesis by down-regulating angiogenic factors as well as reversing the EMT, leading to diminished metastatic spread to the cLNs and lungs. Bottom - Treament of tumors with tipifarnib blocked the activation of HRAS, thus switching off the Hippo pathway and causing YAP1 degradation and down-regulation of AXL expression. This deactivation of HRAS-YAP1-AXL reduced the proliferation and metastasis potential of tumor cells and tumor angiogenesis, leading to diminished metastatic spread to the cLNs and lungs. (This scheme is created with BioRender.com)