Rachel L. Stewart

Clinical significance of the integrin α6β4 in human malignancies

Integrin α6β4 is a cellular adhesion molecule that binds to laminins in the extracellular matrix and nucleates the formation of hemidesmosomes. During carcinoma progression, integrin α6β4 is released from hemidesmosomes, where it can then signal to facilitate multiple aspects of tumor progression including sustaining proliferative signaling, tumor invasion and metastasis, evasion of apoptosis, and stimulation of angiogenesis. The integrin achieves these ends by cooperating with growth factor receptors including EGFR, ErbB-2, and c-Met to amplify downstream pathways such as PI3K, AKT, MAPK, and the Rho family small GTPases. Furthermore, it dramatically alters the transcriptome toward a more invasive phenotype by controlling promoter DNA demethylation of invasion and metastasis-associated proteins, such as S100A4 and autotaxin, and upregulates and activates key tumor-promoting transcription factors such as the NFATs and NF-κB. Expression of integrin α6β4 has been studied in many human malignancies where its overexpression is associated with aggressive behavior and a poor prognosis. This review provides an assessment of integrin α6β4 expression patterns and their prognostic significance in human malignancies, and describes key signaling functions of integrin α6β4 that contribute to tumor progression.

Elevated integrin α6β4 expression is associated with venous invasion and decreased overall survival in non–small cell lung cancer

Lung cancer carries a poor prognosis and is the most common cause of cancer-related death worldwide. The integrin α6β4, a laminin receptor, promotes carcinoma progression in part by cooperating with various growth factor receptors to facilitate invasion and metastasis. In carcinoma cells with mutant TP53, the integrin α6β4 promotes cell survival. TP53 mutations and integrin α6β4 overexpression co-occur in many aggressive malignancies. Because of the high frequency of TP53 mutations in lung squamous cell carcinoma (SCC), we sought to investigate the association of integrin β4 expression with clinicopathologic features and survival in non-small cell lung cancer (NSCLC). We constructed a lung cancer tissue microarray and stained sections for integrin β4 subunit expression using immunohistochemistry. We found that integrin β4 expression is elevated in SCC compared with adenocarcinoma (P<.0001), which was confirmed in external gene expression data sets (P<.0001). We also determined that integrin β4 overexpression associates with the presence of venous invasion (P=.0048) and with reduced overall patient survival (hazard ratio, 1.46; 95% confidence interval, 1.01-2.09; P=.0422). Elevated integrin β4 expression was also shown to associate with reduced overall survival in lung cancer gene expression data sets (hazard ratio, 1.49; 95% confidence interval, 1.31-1.69; P<.0001). Using cBioPortal, we generated a network map demonstrating the 50 most highly altered genes neighboring ITGB4 in SCC, which included laminins, collagens, CD151, genes in the EGFR and PI3K pathways, and other known signaling partners. In conclusion, we demonstrate that integrin β4 is overexpressed in NSCLC where it is an adverse prognostic marker.

Kindlin Binds Migfilin Tandem LIM Domains and Regulates Migfilin Focal Adhesion Localization and Recruitment Dynamics

Background: Migfilin is a focal adhesion protein implicated in control of integrin-mediated cell adhesion, spreading, and migration. Results: Migfilin LIM domains are required for focal adhesion targeting and for binding to kindlin-1 and kindlin-2. Conclusion: Kindlins support migfilin recruitment to focal adhesions and kindlin is important for normal migfilin dynamics in cells. Significance: Migfilin/kindlin interactions are important for regulating subcellular localization. Focal adhesions (FAs), sites of tight adhesion to the extracellular matrix, are composed of clusters of transmembrane integrin adhesion receptors and intracellular proteins that link integrins to the actin cytoskeleton and signaling pathways. Two integrin-binding proteins present in FAs, kindlin-1 and kindlin-2, are important for integrin activation, FA formation, and signaling. Migfilin, originally identified in a yeast two-hybrid screen for kindlin-2-interacting proteins, is a LIM domain-containing adaptor protein found in FAs and implicated in control of cell adhesion, spreading, and migration. By binding filamin, migfilin provides a link between kindlin and the actin cytoskeleton. Here, using a combination of kindlin knockdown, biochemical pulldown assays, fluorescence microscopy, fluorescence resonance energy transfer (FRET), and fluorescence recovery after photobleaching (FRAP), we have established that the C-terminal LIM domains of migfilin dictate its FA localization, shown that these domains mediate an interaction with kindlin in vitro and in cells, and demonstrated that kindlin is important for normal migfilin dynamics in cells. We also show that when the C-terminal LIM domain region is deleted, then the N-terminal filamin-binding region of the protein, which is capable of targeting migfilin to actin-rich stress fibers, is the predominant driver of migfilin localization. Our work details a correlation between migfilin domains that drive kindlin binding and those that drive FA localization as well as a kindlin dependence on migfilin FA recruitment and mobility. We therefore suggest that the kindlin interaction with migfilin LIM domains drives migfilin FA recruitment, localization, and mobility.

S100A4 drives non-small cell lung cancer invasion, associates with poor prognosis, and is effectively targeted by the FDA-approved anti-helminthic agent niclosamide

S100A4 (metastasin-1), a metastasis-associated protein and marker of the epithelial to mesenchymal transition, contributes to several hallmarks of cancer and has been implicated in the progression of several types of cancer. However, the impacts of S100A4 signaling in lung cancer progression and its potential use as a target for therapy in lung cancer have not been properly explored. Using established lung cancer cell lines, we demonstrate that S100A4 knockdown reduces cell proliferation, invasion and three-dimensional invasive growth, while overexpression of S100A4 increases invasive potential. In patient-derived tissues, S100A4 is preferentially elevated in lung adenocarcinoma. This elevation is associated with lymphovascular invasion and decreased overall survival. In addition, depletion of S100A4 by shRNA inhibits NF-κB activity and decreases TNFα-induced MMP9 expression. Furthermore, inhibition of the NF-κB/MMP9 axis decreases lung carcinoma invasive potential. Niclosamide, a reported inhibitor of S100A4, blocks expression and function of S100A4 with a reduction in proliferation, invasion and NF-κB-mediated MMP9 expression. Collectively, this study highlights the importance of the S100A4/NF-κB/MMP9 axis in lung cancer invasion and provides a rationale for targeting S100A4 to combat lung cancer.

Integrin α6β4 Promotes Autocrine Epidermal Growth Factor Receptor (EGFR) Signaling to Stimulate Migration and Invasion toward Hepatocyte Growth Factor (HGF).

Background: Integrin α6β4 is overexpressed in pancreatic cancer and enhances invasion. Results: Integrin α6β4 coordinately up-regulates AREG, EREG, and MMP1 through DNA demethylation and NFAT5 that in turn enhances HGF-mediated invasion. Conclusion: Integrin α6β4 stimulates HGF-dependent invasion through autocrine EGFR signaling. Significance: HGF-stimulated invasion is dependent on autocrine EGFR signaling, thus implicating why EGFR inhibitors are effective in a complex tumor microenvironment. Integrin α6β4 is up-regulated in pancreatic adenocarcinomas where it contributes to carcinoma cell invasion by altering the transcriptome. In this study, we found that integrin α6β4 up-regulates several genes in the epidermal growth factor receptor (EGFR) pathway, including amphiregulin (AREG), epiregulin (EREG), and ectodomain cleavage protease MMP1, which is mediated by promoter demethylation and NFAT5. The correlation of these genes with integrin α6β4 was confirmed in The Cancer Genome Atlas Pancreatic Cancer Database. Based on previous observations that integrin α6β4 cooperates with c-Met in pancreatic cancers, we examined the impact of EGFR signaling on hepatocyte growth factor (HGF)-stimulated migration and invasion. We found that AREG and EREG were required for autocrine EGFR signaling, as knocking down either ligand inhibited HGF-mediated migration and invasion. We further determined that HGF induced secretion of AREG, which is dependent on integrin-growth factor signaling pathways, including MAPK, PI3K, and PKC. Moreover, matrix metalloproteinase activity and integrin α6β4 signaling were required for AREG secretion. Blocking EGFR signaling with EGFR-specific antibodies or an EGFR tyrosine kinase inhibitor hindered HGF-stimulated pancreatic carcinoma cell chemotaxis and invasive growth in three-dimensional culture. Finally, we found that EGFR was phosphorylated in response to HGF stimulation that is dependent on EGFR kinase activity; however, c-Met phosphorylation in response to HGF was unaffected by EGFR signaling. Taken together, these data illustrate that integrin α6β4 stimulates invasion by promoting autocrine EGFR signaling through transcriptional up-regulation of key EGFR family members and by facilitating HGF-stimulated EGFR ligand secretion. These signaling events, in turn, promote pancreatic carcinoma migration and invasion.

Myeloid sarcoma of the breast misdiagnosed as poorly differentiated mammary carcinoma with lobular features.

A 46-year-old Caucasian female was referred to the breast center with complaints of an enlarging, tender breast mass. She had noticed the mass 5 days prior to presentation, and reported that it was accompanied by mild swelling and a burning sensation. On physical exam, she was noted to have a palpable abnormality in the right breast, although examination was somewhat limited by dense breast tissue bilaterally. A mammogram performed 18 months earlier was normal (Fig. 1). The patient’s clinical history was

Principles of Molecular Biology and Oncogenesis

Nucleic acids are macromolecules comprised of chains of nucleotides. In the double helix formed by deoxyribonucleic acid (DNA), purines pair with pyrimidines and are joined together by hydrogen bonds. DNA serves as a template for the transcription of RNA; in a process called translation, RNA provides instructions for the synthesis of proteins. Cancer is a genetic disease. Most cancers are sporadic and result from genetic alterations in somatic cells. Some cancers are inherited, with genetic alterations in germline cells conferring an increased oncogenic risk. Oncogenesis is a complex, dynamic, and often multistep process which is likely dependent on the acquisition of several biological capabilities by somatic cells that result in their independence to external growth signals, insensitivity to external anti-growth signals, indefinite replication, evasion of apoptosis, sustained angiogenesis, activation of tissue invasion and metastasis, reprogramming of energy metabolism, and evasion of host immune response. Knowledge about oncogenic hallmarks has allowed for the establishment and expansion of many areas of personalized cancer care through detection or measurement of molecular biomarkers.

Collagen prolyl 4-hydroxylase 1 is essential for HIF-1α stabilization and TNBC chemoresistance

Collagen prolyl 4-hydroxylase (P4H) expression and collagen hydroxylation in cancer cells are necessary for breast cancer progression. Here, we show that P4H alpha 1 subunit (P4HA1) protein expression is induced in triple-negative breast cancer (TNBC) and HER2 positive breast cancer. By modulating alpha ketoglutarate (α-KG) and succinate levels P4HA1 expression reduces proline hydroxylation on hypoxia-inducible factor (HIF) 1α, enhancing its stability in cancer cells. Activation of the P4HA/HIF-1 axis enhances cancer cell stemness, accompanied by decreased oxidative phosphorylation and reactive oxygen species (ROS) levels. Inhibition of P4HA1 sensitizes TNBC to the chemotherapeutic agent docetaxel and doxorubicin in xenografts and patient-derived models. We also show that increased P4HA1 expression correlates with short relapse-free survival in TNBC patients who received chemotherapy. These results suggest that P4HA1 promotes chemoresistance by modulating HIF-1-dependent cancer cell stemness. Targeting collagen P4H is a promising strategy to inhibit tumor progression and sensitize TNBC to chemotherapeutic agents.Hyperactivation of HIF-1α is crucial in progression of triple-negative breast cancer, but how HIF-1α stability is maintained in a hypoxia-independent manner is unclear. Here, the authors show collagen prolyl-4-hydroylase 1 stabilises HIF-1α and is involved in chemoresistance in TNBC.

Abstract P3-05-06: Evolution of genomic alterations on endocrine therapy and mTOR inhibition in estrogen receptor (ER)-positive breast cancer

Background: Endocrine therapy (ET) and mTOR inhibition are important treatment strategies in ER-positive breast cancer, but no specific genomic alterations reliably predict benefit. Because tumors are tested pretreatment, we hypothesized that this may not capture tumor interaction with therapy. Methods: We studied tumors from protocol NCT00570921 using fulvestrant and everolimus for metastatic ER-positive breast cancer after aromatase inhibitor (AI) failure. DNA from FFPE tumor tissue was subjected to next-generation genomic profiling using the FoundationOne® assay and alterations were then compared between available paired samples: primary/metastatic, before/after everolimus, and upon progression on everolimus. Results: The most common alterations encountered were in the PI3K/AKT/mTOR pathway with increased frequency of PIK3CA in endocrine-sensitive disease but no specific association with everolimus benefit (Massarweh et al, ASCO 2015). One patient with lobular carcinoma relapse on ET and a new contralateral primary, had PIK3CA, CDH1, and MAP3K1 in both tumors with no new alterations detected. Her disease was everolimus sensitive. Another patient with PIK3CA at baseline acquired a CTNNA1 mutation upon relapse with no everolimus benefit. Interestingly, one patient with liver metastasis and complete response to everolimus lasting 3 years had no known alterations reported in the primary tumor but had a PIK3CA mutation in one of two simultaneous biopsies of separate liver lesions. Another patient with liver metastasis and a GATA3 mutation at baseline had response to everolimus lasting 18 months, then developed a PIK3R2_c.1936A>T mutation on progression reported as a variant of unknown significance (VUS). Another patient with metastatic lobular carcinoma to skin and bone had PIK3CA, CDH1, and ERBB2 mutations at baseline, and acquired KRAS and MCL1 amplification on two sequential skin biopsies in the first month on everolimus. She remained on therapy for 1 year. One patient with locally advanced disease and de novo bone metastasis had TP53 and GATA3 mutations at baseline with resistance to multiple chemotherapy and endocrine treatments. Upon progression on AI, her tumor acquired PDGFRA and SMAD4, detected on day 1 biopsy of everolimus treatment. Repeat biopsy on day 28 revealed loss of PDGFRA and SMAD4 with emergence of PIK3CA and MLL2 mutations and loss of STK11. After 1 year on everolimus her tumor progressed and repeat breast biopsy revealed loss of the PIK3CA, STK11, and MLL2 events, with appearance of AKT1 and NF1 mutations. Interestingly, her tumor also acquired ESR1_c.1607T>G, MTOR_c.6104C>T, and NSD1_c.5938G>A mutations, all classified as VUS but were not previously encountered in her course. Further analysis and biologic relevance of these changes will be presented. Conclusion: This small study suggests that ER-positive breast cancer is a dynamic disease with genomic evolution on endocrine therapy and mTOR inhibition. In some patients, this change occurs early on therapy, possibly through clonal selection, but may also be related to tumor heterogeneity. The significance of this change is not fully understood, but study of early on-treatment biopsies may help us better understand tumor response to therapy. Citation Format: Massarweh S, Romond E, Stewart R, Sun J, Chmielecki J, Mehdi M, Black EP. Evolution of genomic alterations on endocrine therapy and mTOR inhibition in estrogen receptor (ER)-positive breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-05-06.

Abstract A44: S100A4 drives the invasive potential of non-small cell lung cancer cells and is targeted by FDA approved drugs

The metastatic nature of advanced non-small cell lung cancer (NSCLC), often associated with therapeutic resistance, accounts for the majority of cancer death with the 5-year survival rate less than 10%. This dismal outcome will remain the same until we gain better understanding of the crucial drivers of the metastatic process and gain the power to effectively target them. S100A4 is a tumor metastasis associated protein and an epithelial to mesenchymal transition (EMT) marker. S100A4 contributes to several hallmarks of cancer such as anti-apoptosis, proliferation and therapeutic resistance, and has been implicated in the progression of different types of cancer, including breast, colon and pancreatic cancer. However, the impact of S100A4 signaling in lung cancer progression and its potential use as a target for therapy in lung cancer has not been properly explored. Here, by using established lung cancer cell lines, we demonstrate that S100A4 is upregulated in a subset of lung cancer cell lines both at the mRNA and protein levels. We further found that inhibition of S100A4 by shRNA in A549 and H460 lung cancer cell lines reduced cell proliferation and decreased 3D invasive growth, while exogenous overexpression of S100A4 in H1299 cells increased invasive potential. Interestingly, we showed that Niclosamide, an FDA-approved anti-tapeworm agent, suppressed S100A4 expression in A549 and H358 cells and led to decreased cell proliferation, invasion and invasive growth. Similarly, we found that a CDC25 inhibitor, NSC95397, disrupts the interaction of S100A4 and non-muscle myosin IIA and also decreased A549 cell invasion. Collectively, these data highlight the importance of S100A4 in lung cancer invasion and metastasis and provide the rationale for targeting S100A4 as a potential agent to combat lung cancer. Citation Format: Min Chen, Rachel Stewart, Teresa Knifley, Brittany L. Carpenter. S100A4 drives the invasive potential of non-small cell lung cancer cells and is targeted by FDA approved drugs. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A44.