Focal adhesion kinase signaling - tumor vulnerabilities and clinical opportunities.

Focal adhesion kinase (FAK; encoded by PTK2) was discovered over 30 years ago as a cytoplasmic protein tyrosine kinase that is localized to cell adhesion sites, where it is activated by integrin receptor binding to extracellular matrix proteins. FAK is ubiquitously expressed and functions as a signaling scaffold for a variety of proteins at adhesions and in the cell cytoplasm, and with transcription factors in the nucleus. FAK expression and intrinsic activity are essential for mouse development, with molecular connections to cell motility, cell survival and gene expression. Notably, elevated FAK tyrosine phosphorylation is common in tumors, including pancreatic and ovarian cancers, where it is associated with decreased survival. Small molecule and orally available FAK inhibitors show on-target inhibition in tumor and stromal cells with effects on chemotherapy resistance, stromal fibrosis and tumor microenvironment immune function. Herein, we discuss recent insights regarding mechanisms of FAK activation and signaling, its roles as a cytoplasmic and nuclear scaffold, and the tumor-intrinsic and -extrinsic effects of FAK inhibitors. We also discuss results from ongoing and advanced clinical trials targeting FAK in low- and high-grade serous ovarian cancers, where FAK acts as a master regulator of drug resistance. Although FAK is not known to be mutationally activated, preventing FAK activity has revealed multiple tumor vulnerabilities that support expanding clinical combinatorial targeting possibilities.

Tumor FAK orchestrates immunosuppression in ovarian cancer via the CD155/TIGIT axis.

High-grade serous ovarian cancer (HGSOC) is a lethal malignancy characterized by an immunosuppressive tumor microenvironment containing few tumor infiltrating lymphocytes (TILs) and an insensitivity to checkpoint inhibitor immunotherapies. Gains in the PTK2 gene encoding focal adhesion kinase (FAK) at Chr8 q24.3 occur in ∼70% of HGSOC tumors, and elevated FAK messenger RNA (mRNA) levels are associated with poor patient survival. Herein, we show that active FAK, phosphorylated at tyrosine-576 within catalytic domain, is significantly increased in late-stage HGSOC tumors. Active FAK costained with CD155, a checkpoint receptor ligand for TIGIT (T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domains), in HGSOC tumors and a selective association between FAK and TIGIT checkpoint ligands were supported by patient transcriptomic database analysis. HGSOC tumors with high FAK expression were associated with low CD3 mRNA levels. Accordingly, late-stage tumors showed elevated active FAK staining and significantly lower levels of CD3+ TILs. Using the KMF (Kras, Myc, FAK) syngeneic ovarian tumor model containing spontaneous PTK2 (FAK) gene gains, the effects of tumor intrinsic genetic or oral small molecule FAK inhibitior (FAKi; VS-4718) were evaluated in vivo. Blocking FAK activity decreased tumor burden, suppressed ascites KMF-associated CD155 levels, and increased peritoneal TILs. The combination of FAKi with blocking TIGIT antibody (1B4) maintained elevated TIL levels and reduced TIGIT+ T regulatory cell levels, prolonged host survival, increased CXCL13 levels, and led to the formation of omental tertiary lymphoid structures. Collectively, our studies support FAK and TIGIT targeting as a rationale immunotherapy combination for HGSOC.

Autophagy gene haploinsufficiency drives chromosome instability, increases migration, and promotes early ovarian tumors.

Autophagy, particularly with BECN1, has paradoxically been highlighted as tumor promoting in Ras-driven cancers, but potentially tumor suppressing in breast and ovarian cancers. However, studying the specific role of BECN1 at the genetic level is complicated due to its genomic proximity to BRCA1 on both human (chromosome 17) and murine (chromosome 11) genomes. In human breast and ovarian cancers, the monoallelic deletion of these genes is often co-occurring. To investigate the potential tumor suppressor roles of two of the most commonly deleted autophagy genes in ovarian cancer, BECN1 and MAP1LC3B were knocked-down in atypical (BECN1+/+ and MAP1LC3B+/+) ovarian cancer cells. Ultra-performance liquid chromatography mass-spectrometry metabolomics revealed reduced levels of acetyl-CoA which corresponded with elevated levels of glycerophospholipids and sphingolipids. Migration rates of ovarian cancer cells were increased upon autophagy gene knockdown. Genomic instability was increased, resulting in copy-number alteration patterns which mimicked high grade serous ovarian cancer. We further investigated the causal role of Becn1 haploinsufficiency for oncogenesis in a MISIIR SV40 large T antigen driven spontaneous ovarian cancer mouse model. Tumors were evident earlier among the Becn1+/- mice, and this correlated with an increase in copy-number alterations per chromosome in the Becn1+/- tumors. The results support monoallelic loss of BECN1 as permissive for tumor initiation and potentiating for genomic instability in ovarian cancer.

Underlying Subwavelength Aperture Architecture Drives the Optical Properties of Microcavity Surface Plasmon Resonance Sensors.

Microcavity surface plasmon resonance sensors (MSPRSs) develop out of the classic surface plasmon resonance technologies and aim at producing novel lab-on-a-chip devices. MSPRSs generate a series of spectral resonances sensitive to minute changes in the refractive index. Related sensitivity studies and biosensing applications are published elsewhere. The goal of this work is to test the hypothesis that MSPRS resonances are standing surface plasmon waves excited at the surface of the sensor that decay back into propagating photons. Their optical properties (mean wavelength, peak width, and peak intensity) appear highly dependent on the internal morphology of the sensor and the underlying subwavelength aperture architecture in particular. Numerous optical experiments were designed to investigate trends that confirm this hypothesis. An extensive study of prior works was supportive of our findings and interpretations. A complete understanding of those mechanisms and parameters driving the formations of the MSPRS resonances would allow further improvement in sensor sensitivity, reliability, and manufacturability.

PFKFB4 Promotes Breast Cancer Metastasis via Induction of Hyaluronan Production in a p38-Dependent Manner.

BACKGROUND/AIMS: The bi-functional enzyme 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase-4 (PFKFB4) is highly expressed in many types of cancer and its requirement for tumor survival has been demonstrated in glioma, lung, and prostate cancers. However, whether PFKFB4 plays a role in the tumor metastasis remains uncertain. This study explores the role of PFKFB4 in tumor metastasis and its underlying mechanisms in breast cancer cells. METHODS: The expression of PFKFB4 was first analyzed using the Cancer Genome Atlas (TCGA) dataset, and confirmed by immunohistochemical staining of tissue microarray and breast cancer tissues from patient samples. Gain- and loss-of- function approaches were used to investigate the effects of PFKFB4 on breast cancer cell migration in vitro. Orthotopic xenograft model and experimental metastasis model were used to assess the effects of PFKFB4 on breast cancer cell metastasis in vivo. ELISA and immunofluorescence staining were used to examine HA production. Quantitative RT-PCR and western blotting were used to explore the mRNA and protein levels of HAS2, respectively. RESULTS: We found that PFKFB4 enhances the migration/invasiveness of breast cancer cells in vitro as well as in vivo. Notably, the effects of PFKFB4 on migration are mediated by induction of HAS2 expression and HA production. Moreover, PFKFB4-induced HAS2 up-regulation depends upon the activation of p38 signaling. CONCLUSION: PFKFB4 promotes the metastasis of breast cancer cells via induction of HAS2 expression and HA production in a p38-dependent manner. Therefore, the PFKFB4/p38/HAS2 signaling pathway may serve as a potential therapeutic target for metastatic breast cancer.

The Ras-related protein, Rap1A, mediates thrombin-stimulated, integrin-dependent glioblastoma cell proliferation and tumor growth.

Rap1 is a Ras family GTPase with a well documented role in ERK/MAP kinase signaling and integrin activation. Stimulation of the G-protein-coupled receptor PAR-1 with thrombin in human 1321N1 glioblastoma cells led to a robust increase in Rap1 activation. This response was sustained for up to 6 h and mediated through RhoA and phospholipase D (PLD). Thrombin treatment also induced a 5-fold increase in cell adhesion to fibronectin, which was blocked by down-regulating PLD or Rap1A or by treatment with a ß1 integrin neutralizing antibody. In addition, thrombin treatment led to increases in phospho-focal adhesion kinase (tyrosine 397), ERK1/2 phosphorylation and cell proliferation, which were significantly inhibited in cells treated with ß1 integrin antibody or Rap1A siRNA. To assess the role of Rap1A in tumor formation in vivo, we compared growth of 1321N1 cells stably expressing control, Rap1A or Rap1B shRNA in a mouse xenograft model. Deletion of Rap1A, but not of Rap1B, reduced tumor mass by >70% relative to control. Similar observations were made with U373MG glioblastoma cells in which Rap1A was down-regulated. Collectively, these findings implicate a Rap1A/ß1 integrin pathway, activated downstream of G-protein-coupled receptor stimulation and RhoA, in glioblastoma cell proliferation. Moreover, our data demonstrate a critical role for Rap1A in glioblastoma tumor growth in vivo.

Caspase-8 isoform 6 promotes death effector filament formation independent of microtubules.

Caspase-8 can trigger cell death following prodomain-mediated recruitment to the 'death-inducing signaling complex.' The prodomain consists of two death effector domain (DED) motifs that undergo homotypic interactions within the cell. Aside from mediating recruitment of procaspase-8, the prodomains have also been implicated in regulating cell survival, proliferation, death, senescence, differentiation, and substrate attachment. Here, we perform the initial characterization of a novel isoform of caspase-8, designated caspase-8 isoform 6 (Casp-8.6), which encodes both prodomain DEDs followed by a unique C-terminal tail. Casp-8.6 is detected in cells of the hematopoietic compartment as well as several other tissues. When Casp-8.6 expression is reconstituted in caspase-8-deficient cells, Casp-8.6 does not significantly impact cellular proliferation, contrasting with our previous results using a domain-defined 'DED-only' construct that lacks the C-terminal tail. Like the DED-only construct, Casp-8.6 also robustly forms 'death effector' filaments, but in contrast to the DED construct, it does not exhibit a dependence upon intact microtubules to scaffold filament formation. Both types of death effector filaments promote apoptosis when expressed in the presence of full length caspase-8 (isoform 1). Together, the results implicate Casp-8.6 as a new physiological modulator of apoptosis.

Potentiation of neuroblastoma metastasis by loss of caspase-8.

Neuroblastoma, the most common paediatric solid tumour, arises from defective neural crest cells. Genetic alterations occur frequently in the most aggressive neuroblastomas. In particular, deletion or suppression of the proapoptotic enzyme caspase-8 is common in malignant, disseminated disease, although the effect of this loss on disease progression is unclear. Here we show that suppression of caspase-8 expression occurs during the establishment of neuroblastoma metastases in vivo, and that reconstitution of caspase-8 expression in deficient neuroblastoma cells suppressed their metastases. Caspase-8 status was not a predictor of primary tumour growth; rather, caspase-8 selectively potentiated apoptosis in neuroblastoma cells invading the collagenous stroma at the tumour margin. Apoptosis was initiated by unligated integrins by means of a process known as integrin-mediated death. Loss of caspase-8 or integrin rendered these cells refractory to integrin-mediated death, allowed cellular survival in the stromal microenvironment, and promoted metastases. These findings define caspase-8 as a metastasis suppressor gene that, together with integrins, regulates the survival and invasive capacity of neuroblastoma cells.

Nonsense-mediated RNA decay: an emerging modulator of malignancy.

Nonsense-mediated RNA decay (NMD) is a highly conserved RNA turnover pathway that selectively degrades RNAs harbouring truncating mutations that prematurely terminate translation, including nonsense, frameshift and some splice-site mutations. Recent studies show that NMD shapes the mutational landscape of tumours by selecting for mutations that tend to downregulate the expression of tumour suppressor genes but not oncogenes. This suggests that NMD can benefit tumours, a notion further supported by the finding that mRNAs encoding immunogenic neoantigen peptides are typically targeted for decay by NMD. Together, this raises the possibility that NMD-inhibitory therapy could be of therapeutic benefit against many tumour types, including those with a high load of neoantigen-generating mutations. Complicating this scenario is the evidence that NMD can also be detrimental for many tumour types, and consequently tumours often have perturbed NMD. NMD may suppress tumour generation and progression by degrading subsets of specific normal mRNAs, including those encoding stress-response proteins, signalling factors and other proteins beneficial for tumours, as well as pro-tumour non-coding RNAs. Together, these findings suggest that NMD-modulatory therapy has the potential to provide widespread therapeutic benefit against diverse tumour types. However, whether NMD should be stimulated or repressed requires careful analysis of the tumour to be treated.

The marine lipopeptide somocystinamide A triggers apoptosis via caspase 8.

Screening for novel anticancer drugs in chemical libraries isolated from marine organisms, we identified the lipopeptide somocystinamide A (ScA) as a pluripotent inhibitor of angiogenesis and tumor cell proliferation. The antiproliferative activity was largely attributable to induction of programmed cell death. Sensitivity to ScA was significantly increased among cells expressing caspase 8, whereas siRNA knockdown of caspase 8 increased survival after exposure to ScA. ScA rapidly and efficiently partitioned into liposomes while retaining full antiproliferative activity. Consistent with the induction of apoptosis via the lipid compartment, we noted accumulation and aggregation of ceramide in treated cells and subsequent colocalization with caspase 8. Angiogenic endothelial cells were extremely sensitive to ScA. Picomolar concentrations of ScA disrupted proliferation and endothelial tubule formation in vitro. Systemic treatment of zebrafish or local treatment of the chick chorioallantoic membrane with ScA resulted in dose-dependent inhibition of angiogenesis, whereas topical treatment blocked tumor growth among caspase-8-expressing tumors. Together, the results reveal an unexpected mechanism of action for this unique lipopeptide and suggest future development of this and similar agents as antiangiogenesis and anticancer drugs.

Association of Copy Number Variation Signature and Survival in Patients With Serous Ovarian Cancer.

Importance: Tailoring therapeutic regimens to individual patients with ovarian cancer is informed by severity of disease using a variety of clinicopathologic indicators. Although DNA repair variations are increasingly used for therapy selection in ovarian cancer, molecular features are not widely used for general assessment of patient prognosis and disease severity. Objective: To distill a highly dynamic characteristic, signature of copy number variations (CNV), into a risk score that could be easily validated analytically or repurposed for use given existing US Food and Drug Administration (FDA)-approved multigene assays. Design, Setting, and Participants: This genetic association study used the Cancer Genome Atlas Ovarian Cancer database to assess for genome-wide survival associations agnostic to gene function. Regions enriched for significant associations were compared to associations from scrambled data. CNV associations were condensed into a risk score, which was internally validated using bootstrapping. The participants were patients with serous ovarian cancer (stages I-IV) diagnosed from 1992 to 2013. Statistical analysis was performed from April to July 2020. Main Outcomes and Measures: Overall survival (OS). Results: Among 564 patients with serous ovarian cancer, the mean (SD) age was 59.7 (11.5) years; 34 (6%) identified as Black or African American. A total of 13 genome regions, comprising 14 alterations, were identified as significantly risk associated. Composite risk score was independent of total CNV burden, total mutational burden, BRCA status, and open-source genome-wide DNA repair deficiency signatures. Binned terciles yielded high-, standard-, and low-risk groups with respective median OS estimates of 2.9 (95% CI, 2.3-3.2) years, 4.1 (95% CI, 3.7-4.8) years, and 5.7 (95% CI, 4.7-7.4) years, respectively (P < .001). Associated 5-year survival estimates in each tercile were 15% (95% CI, 10%-22%), 36% (95% CI, 29%-46%), and 53% (95% CI, 45%-62%). The risk score had more discriminatory ability to prognosticate OS than age, clinical stage, grade, and race combined, and was strongly additive to significant clinical features (P < .001). Simulated adaptation of FDA-approved assays showed similar performance. Gene ontology analyses of identified regions showed an enrichment for regulatory miRNAs and protein kinase regulators. Conclusions and Relevance: This study found that a CNV-based risk score is independent to and stronger than current or near-future ovarian cancer genomic biomarkers to prognosticate OS. CNV regions identified were not strongly associated with canonical ovarian cancer biological pathways, identifying candidates for future mechanistic investigations. External validation of the CNV risk score, especially in concert with more extensive clinical features, could be pursued via existing FDA-approved assays.

Targeting FAK in anticancer combination therapies.

Focal adhesion kinase (FAK) is both a non-receptor tyrosine kinase and an adaptor protein that primarily regulates adhesion signalling and cell migration, but FAK can also promote cell survival in response to stress. FAK is commonly overexpressed in cancer and is considered a high-value druggable target, with multiple FAK inhibitors currently in development. Evidence suggests that in the clinical setting, FAK targeting will be most effective in combination with other agents so as to reverse failure of chemotherapies or targeted therapies and enhance efficacy of immune-based treatments of solid tumours. Here, we discuss the recent preclinical evidence that implicates FAK in anticancer therapeutic resistance, leading to the view that FAK inhibitors will have their greatest utility as combination therapies in selected patient populations.

Src-mediated coupling of focal adhesion kinase to integrin alpha(v)beta5 in vascular endothelial growth factor signaling.

Vascular endothelial growth factor (VEGF) promotes vascular permeability (VP) and neovascularization, and is required for development. We find that VEGF-stimulated Src activity in chick embryo blood vessels induces the coupling of focal adhesion kinase (FAK) to integrin alpha(v)beta5, a critical event in VEGF-mediated signaling and biological responsiveness. In contrast, FAK is constitutively associated with beta1 and beta3 integrins in the presence or absence of growth factors. In cultured endothelial cells, VEGF, but not basic fibroblast growth factor, promotes the Src-mediated phosphorylation of FAK on tyrosine 861, which contributes to the formation of a FAK/alpha(v)beta5 signaling complex. Moreover, formation of this FAK/alpha(v)beta5 complex is significantly reduced in pp60c-src-deficient mice. Supporting these results, mice deficient in either pp60c-src or integrin beta5, but not integrin beta3, have a reduced VP response to VEGF. This FAK/alpha(v)beta5 complex was also detected in epidermal growth factor-stimulated epithelial cells, suggesting a function for this complex outside the endothelium. Our findings indicate that Src can coordinate specific growth factor and extracellular matrix inputs by recruiting integrin alpha(v)beta5 into a FAK-containing signaling complex during growth factor-mediated biological responses.

Rgnef promotes ovarian tumor progression and confers protection from oxidative stress.

Ovarian cancer is the fifth-leading cause of cancer death among women. The dissemination of ovarian tumors and growth as spheroids accompanies late-stage disease. In cell culture, ovarian tumor cell spheroids can exhibit elevated resistance to environmental stressors, such as reactive oxygen species. Homeostatic balance of the antioxidant response is a protective mechanism that prevents anoikis, a form of programmed cell death. Signaling pathways activated by integrin receptors suppress anoikis. Rgnef (ARHGEF28/p190RhoGEF) is a guanine nucleotide exchange factor that is activated downstream of integrins. We find that Rgnef protein levels are elevated in late-stage serous ovarian cancer, high Rgnef mRNA levels are associated with decreased progression-free and overall survival, and genomic ARHGEF28 loss is associated with increased patient survival. Using transgenic and transplantable Rgnef knockout mouse models, we find that Rgnef is essential for supporting three-dimensional ovarian spheroid formation in vitro and tumor growth in mice. Using RNA-sequencing and bioinformatic analyses, we identify a conserved Rgnef-supported anti-oxidant gene signature including Gpx4, Nqo1, and Gsta4; common targets of the NF-kB transcription factor. Antioxidant treatment enhanced growth of Rgnef-knockout spheroids and Rgnef re-expression facilitated NF-κB-dependent tumorsphere survival. These studies reveal a new role for Rgnef in ovarian cancer to facilitate NF-κB-mediated gene expression protecting cells from oxidative stress.

FAK activity sustains intrinsic and acquired ovarian cancer resistance to platinum chemotherapy.

Gene copy number alterations, tumor cell stemness, and the development of platinum chemotherapy resistance contribute to high-grade serous ovarian cancer (HGSOC) recurrence. Stem phenotypes involving Wnt-ß-catenin, aldehyde dehydrogenase activities, intrinsic platinum resistance, and tumorsphere formation are here associated with spontaneous gains in Kras, Myc and FAK (KMF) genes in a new aggressive murine model of ovarian cancer. Adhesion-independent FAK signaling sustained KMF and human tumorsphere proliferation as well as resistance to cisplatin cytotoxicity. Platinum-resistant tumorspheres can acquire a dependence on FAK for growth. Accordingly, increased FAK tyrosine phosphorylation was observed within HGSOC patient tumors surviving neo-adjuvant chemotherapy. Combining a FAK inhibitor with platinum overcame chemoresistance and triggered cell apoptosis. FAK transcriptomic analyses across knockout and reconstituted cells identified 135 targets, elevated in HGSOC, that were regulated by FAK activity and ß-catenin including Myc, pluripotency and DNA repair genes. These studies reveal an oncogenic FAK signaling role supporting chemoresistance.

Does integrin-mediated cell death confer tissue tropism in metastasis?

To develop metastatic capability, tumor cells must evolve the capacity to survive in novel microenvironments. Recently, we showed that metastasis of neuroblastoma cells is enhanced by loss of caspase-8, an event that occurs frequently in this malignancy. In poorly metastatic cells, unligated integrins were found to trigger activation of caspase-8, providing a selective pressure to promote its attenuation and thereby increased survival in foreign adhesive environments. Our findings suggest one mechanism by which the organotropism of metastatic cancer cells can arise.

CD44ICD promotes breast cancer stemness via PFKFB4-mediated glucose metabolism.

CD44 is a single-pass cell surface glycoprotein that is distinguished as the first molecule used to identify cancer stem cells in solid tumors based on its expression. In this regard, the CD44high cell population demonstrates not only the ability to regenerate a heterogeneous tumor, but also the ability to self-regenerate when transplanted into immune-deficient mice. However, the exact role of CD44 in cancer stem cells remains unclear in part because CD44 exists in various isoforms due to alternative splicing. Methods: Gain- and loss-of-function methods in different models were used to investigate the effects of CD44 on breast cancer stemness. Cancer stemness was analyzed by detecting SOX2, OCT4 and NANOG expression, ALDH activity, side population (SP) and sphere formation. Glucose consumption, lactate secretion and reactive oxygen species (ROS) levels were detected to assess glycolysis. Western blot, immunohistochemical staining, ELISA and TCGA dataset analysis were performed to determine the association of CD44ICD and PFKFB4 with clinical cases. A PFKFB4 inhibitor, 5MPN, was used in a xenograft model to inhibit breast cancer development. Results: In this report, we found that the shortest CD44 isoform (CD44s) inhibits breast cancer stemness, whereas the cleaved product of CD44 (CD44ICD) promotes breast cancer stemness. Furthermore, CD44ICD interacts with CREB and binds to the promoter region of PFKFB4, thereby regulating PFKFB4 transcription and expression. The resultant PFKFB4 expression facilitates the glycolysis pathway (vis-à-vis oxidative phosphorylation) and promotes stemness of breast cancer. In addition, we found that CD44ICD and PFKFB4 expressions are generally up-regulated in the tumor portion of breast cancer patient samples. Most importantly, we found that 5MPN (a selective inhibitor of PFKFB4) suppresses CD44ICD-induced tumor development. Conclusion: CD44ICD promotes breast cancer stemness via PFKFB4-mediated glycolysis, and therapies that target PFKFB4 (e.g., 5MPN therapy) may lead to improved outcomes for cancer patients.

Cell survival in a three-dimensional matrix.

Integrin-mediated adhesion acts as a pluripotent mediator of cell signaling, triggering many pathways that promote proliferation and permit them to resist exogenous proapototic insults. To date, most studies have focused on apoptosis among cells adherent to rigid tissue-culture plastic substrates that tends to maximize integrin survival signaling. The physiological interpretation of such studies remains unclear. Here we describe methods to study integrin-mediated cell survival using matched cell populations that differ only in integrin expression, using a three-dimensional (3D) extracellular matrix culture. The preparation of appropriate cell types as well as the use of 3D collagen and fibrin gels is described. Methods to assess apoptosis and their application in the model are detailed. These techniques will offer an opportunity to study cell survival in the context of a non-rigid 3D extracellular matrix.