Sarmishtha De

Molecular Pathway for Cancer Metastasis to Bone

The molecular mechanism leading to the cancer metastasis to bone is poorly understood but yet determines prognosis and therapy. Here, we define a new molecular pathway that may account for the extraordinarily high osteotropism of prostate cancer. By using SPARC (secreted protein, acidic and rich in cysteine)-deficient mice and recombinant SPARC, we demonstrated that SPARC selectively supports the migration of highly metastatic relative to less metastatic prostate cancer cell lines to bone. Increased migration to SPARC can be traced to the activation of integrins αVβ3 and αVβ5 on tumor cells. Such activation is induced by an autocrine vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR)-2 loop on the tumor cells, which also supports the growth and proliferation of prostate cancer cells. A consequence of SPARC recognition by αVβ5 is enhanced VEGF production. Thus, prostate cancer cells expressing VEGF/VEGFR-2 will activate αVβ3 and αVβ5 on their surface and use these integrins to migrate toward SPARC in bone. Within the bone environment, SPARC engagement of these integrins will stimulate growth of the tumor and further production of VEGF to support neoangiogenesis, thereby favoring the development of the metastatic tumor. Supporting this model, activated integrins were found to colocalize with VEGFR-2 in tissue samples of metastatic prostate tumors from patients.

Prostate cancer specific integrin αvβ3 modulates bone metastatic growth and tissue remodeling

The management of pain and morbidity due to the spreading and growth of cancer within bone remains to be a paramount problem in clinical care. Cancer cells actively transform bone, however, the molecular requirements and mechanisms of this process remain unclear. This study shows that functional modulation of the αvβ3 integrin receptor in prostate cancer cells is required for progression within bone and determines tumor-induced bone tissue transformation. Using histology and quantitative microCT analysis, we show that αvβ3 integrin is required not only for tumor growth within the bone but for tumor-induced bone gain, a response resembling bone lesions in prostate cancer patients. Expression of normal, fully functional αvβ3 enabled tumor growth in bone (incidence: 4/4), whereas αvβ3 (−), inactive or constitutively active mutants of αvβ3 did not (incidence: 0/4, 0/6 and 1/7, respectively) within a 35-day-period. This response appeared to be bone-specific in comparison to the subcutis where tumor incidence was greater than 60% for all groups. Interestingly, bone residing prostate cancer cells expressing normal or dis-regulated αvβ3 (either inactive of constitutively active), but not those lacking β3 promoted bone gain or afforded protection from bone loss in the presence or absence of histologically detectable tumor 35 days following implantation. As bone is replete with ligands for β3 integrin, we next demonstrated that αvβ3 integrin activation on tumor cells is essential for the recognition of key bone-specific matrix proteins. As a result, prostate cancer cells expressing fully functional but not dis-regulated αvβ3 integrin are able to control their own adherence and migration to bone matrix, functions that facilitate tumor growth and control bone lesion development.

Metastatic properties of prostate cancer cells are controlled by VEGF

Mechanisms of metastasis, the major complication of prostate cancer, are poorly understood. In this study, we define molecular mechanisms that may contribute to the highly invasive potential of prostate cancer cells. Vascular endothelial growth factor (VEGF), its receptors (VEGFRs), and α5β1 integrin were expressed by prostate cancer cells in vitro and by prostate tumors in vivo, and their expression was elevated at sites of bone metastasis compared to original prostate tumor. VEGF, through interaction with its receptors, regulated adhesive and migratory properties of the cancer cells. Specifically, the highly metastatic prostate cancer cell subline LNCaP-C4-2 showed a decreased adhesive but an enhanced migratory response to fibronectin, a ligand for α5β1 integrin, compared to its nonmetastatic counterpart. A similar pattern was also observed when bone sialoprotein was used as a ligand in migration assays. Increased migration of metastatic prostate cancer cells to fibronectin and bone sialoprotein was regulated by VEGF via VEGFR-2. Tumor suppressor PTEN was involved in control of VEGF/VEGFR-2 stimulated prostate cancer cell adhesion as well as proliferation.

Erlotinib protects against LPS-induced Endotoxicity because TLR4 needs EGFR to signal

Significance The activation of nuclear factor κB (NFκB) in the normal inflammatory response is rapidly down regulated, whereas constitutive NFκB activation is a hallmark of cancer. We now reveal cross signaling between EGF receptor (EGFR) and Toll-like receptor 4 (TLR4). NFκB activation in response to EGF requires, in addition to EGFR, TLR4 and two downstream proteins. Conversely, EGFR is required for TLR4-mediated activation of NFκB in response to lipopolysaccharide (LPS). The LYN proto-oncogene (LYN) is required for NFκB activation in response to either ligand. In mice, the EGFR inhibitor erlotinib greatly reduces both cytokine expression and endotoxicity in response to LPS, suggesting that EGFR inhibitors may find use in treating septic shock. Several components of the canonical pathway of response to lipopolysaccharide (LPS) are required for the EGF-dependent activation of NFκB. Conversely, the ability of Toll-like Receptor 4 (TLR4) to activate NFκB in response to LPS is impaired by down regulating EGF receptor (EGFR) expression or by using the EGFR inhibitor erlotinib. The LYN proto-oncogene (LYN) is required for signaling in both directions. LYN binds to the EGFR upon LPS stimulation, and erlotinib impairs this association. In mice, erlotinib blocks the LPS-induced expression of tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) and ameliorates LPS-induced endotoxity, revealing that EGFR is essential for LPS-induced signaling in vivo.

EGF receptor uses SOS1 to drive constitutive activation of NFκB in cancer cells.

Significance In normal cells, quiescent nuclear factor κB (NFκB) is activated by inflammatory stimuli. In most cancers, the abnormal constitutive activation of NFκB contributes to malignant progression and resistance to therapy. Overexpression or constitutive activation of the EGF receptor (EGFR) in many cancers contributes to their proliferation and survival. We find that the constitutive activation of NFκB in several cancer cell lines is decreased by EGFR knockdown or by the EGFR inhibitor erlotinib. We used insertional mutagenesis to find that overexpression of Son of Sevenless 1 (SOS1), a component of EGF-dependent pathways that facilitate cell growth and survival, causes erlotinib resistance and increases NFκB activation. SOS1 is required for EGF-dependent activation of NFκB but its GDP–GTP exchange activity is not, revealing a novel function for this protein. Activation of nuclear factor κB (NFκB) is a central event in the responses of normal cells to inflammatory signals, and the abnormal constitutive activation of NFκB is important for the survival of most cancer cells. In nonmalignant human cells, EGF stimulates robust activation of NFκB. The kinase activity of the EGF receptor (EGFR) is required, because the potent and specific inhibitor erlotinib blocks the response. Down-regulating EGFR expression or inhibiting EGFR with erlotinib impairs constitutive NFκB activation in several different types of cancer cells and, conversely, increased activation of NFκB leads to erlotinib resistance in these cells. We conclude that EGF is an important mediator of NFκB activation in cancer cells. To explore the mechanism, we selected an erlotinib-resistant cell line in which the guanine nucleotide exchange factor Son of Sevenless 1 (SOS1), well known to be important for EGF-dependent signaling to MAP kinases, is overexpressed. Increased expression of SOS1 increases NFκB activation in several different types of cancer cells, and ablation of SOS1 inhibits EGF-induced NFκB activation in these cells, indicating that SOS1 is a functional component of the pathway connecting EGFR to NFκB activation. Importantly, the guanine nucleotide exchange activity of SOS1 is not required for NFκB activation.

The FACT inhibitor CBL0137 synergizes with cisplatin in small cell lung cancer by increasing NOTCH1 expression and targeting tumor-initiating cells

Traditional treatments of small-cell lung cancer (SCLC) with cisplatin, a standard-of-care therapy, spare the tumor-initiating cells (TIC) that mediate drug resistance. Here we report a novel therapeutic strategy that preferentially targets TICs in SCLC, in which cisplatin is combined with CBL0137, an inhibitor of the histone chaperone facilitates chromatin transcription (FACT), which is highly expressed in TICs. Combination of cisplatin and CBL0137 killed patient-derived and murine SCLC cell lines synergistically. In response to CBL0137 alone, TICs were more sensitive than non-TICs, in part, because CBL0137 increased expression of the tumor suppressor NOTCH1 by abrogating the binding of negative regulator SP3 to the NOTCH1 promoter, and in part because treatment decreased the high expression of stem cell transcription factors. The combination of cisplatin and CBL0137 greatly reduced the growth of a patient-derived xenograft in mice and also the growth of a syngeneic mouse SCLC tumor. Thus, CBL0137 can be a highly effective drug against SCLC, especially in combination with cisplatin.Significance: These findings reveal a novel therapeutic regimen for SCLC, combining cisplatin with an inhibitor that preferentially targets tumor-initiating cells. Cancer Res; 78(9); 2396-406. ©2018 AACR.

Abstract 1944: NFκB: Activation by EGF requires TLR4 and activation by LPS requires EGFR

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA In normal cells, quiescent NFκB is activated by inflammatory stimuli, whereas the abnormal constitutive activation of NFκB is important for the survival of most cancer cells. NFκB activation is regulated by several different pathways. Recently we showed that EGFR is an important mediator of NFκB activation, in both non-malignant and malignant cells. We now provide additional mechanistic insight into this pathway by further investigating whether signaling to NFκB in response to EGF depends solely on EGFR, or involves another receptor known to activate NFκB. Knockdown experiments revealed that TLR4 and two downstream components in the canonical TLR4 pathway, MYD88 and TAK1, are all required for EGF-dependent NFκB activation. Conversely, the ability of TLR4 to activate NFκB in response to LPS was impaired by down regulating EGFR expression or by use of the EGFR inhibitor erlotinib. PP2, a general inhibitor of SRC-family kinases, blocked EGF-induced NFκB activation, and the SRC family member LYN became bound to EGFR upon LPS stimulation. Erlotinib blocked this LPS-dependent association, indicating a key role for LYN in EGFR-TLR4 cross signaling. In mice, erlotinib blocked the LPS-induced, NFκB-dependent expression of TNFα and IL-6, indicating that EGFR is essential for LPS-induced NFκB activation in vivo. Citation Format: Sarmishtha De, Hao Zhou, Xiaoxia Li, George Stark. NFκB: Activation by EGF requires TLR4 and activation by LPS requires EGFR. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1944. doi:10.1158/1538-7445.AM2015-1944