Inhibition of Non-Small Cell Lung Cancer Cells by Oxy210, an Oxysterol-Derivative that Antagonizes TGFβ and Hedgehog Signaling

Abstract

Non-Small Cell Lung Cancer (NSCLC) is a common malignancy and leading cause of death by cancer. Metastasis and drug resistance are serious clinical problems encountered in NSCLC therapy. Aberrant activation of the Transforming Growth Factor beta (TGFβ) and Hedgehog (Hh) signal transduction cascades often associate with poor prognosis and aggressive disease progression in NSCLC, as these signals can drive cell proliferation, angiogenesis, metastasis, immune evasion and emergence of drug resistance. Therefore, simultaneous inhibition of TGFβ and Hh signaling, by a single agent, or in combination with other drugs, could yield therapeutic benefits in NSCLC and other cancers. In the current study, we report on the biological and pharmacological evaluation of Oxy210, an oxysterol-based dual inhibitor of TGFβ and Hh signaling. In NSCLC cells, Oxy210 inhibits proliferation, epithelial-mesenchymal transition (EMT) and invasive activity. Combining Oxy210 with Carboplatin (CP) increases the anti-proliferative response to CP and inhibits TGFβ-induced resistance to CP in A549 NSCLC cells. In addition, Oxy210 displays encouraging drug-like properties, including chemical scalability, metabolic stability and oral bioavailability in mice. Unlike other known inhibitors, Oxy210 antagonizes TGFβ and Hh signaling independently of TGFβ receptor kinase inhibition and downstream of Smoothened, respectively.

Identification of anti-severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) oxysterol derivatives in vitro

“…(E, F) Inhibitory effects toward TGFβ and Hh signaling. NIH3T3 cells pretreated with or without Oxy210 or Oxy232 for 2 h were stimulated with 20 ng/mL TGFβ (E) or with conditioned medium from CAPAN-1 human pancreatic tumor cells that contain Shh (F) [15, 16] in the presence or absence of the compounds. Cellular mRNAs were extracted to quantify a TGFβ-downstream gene, connective tissue growth factor (CTGF) (E), a Hh target gene, Gli1 (F), and Oaz1 for normalization of CTGF and Gli1 (E, F).…”

“…We previously reported that Oxy210 inhbited Hedgehog (Hh) and transforming growth factor β (TGFβ) signalings in fibroblastic cells and tumor cells [16]. In contrast, Oxy232, a close structural analogue of Oxy210, is devoid of significant TGFβ inhibitory properties (Figure 2E), but retains inhibitory activity toward Hh signaling (Figure 2F), suggesting that inhibition of TGFβ signaling is not resposible for the anti-SARS-CoV-2 activity.…”

“…Given its higher anti-SARS-CoV-2 potency compared to the natural oxysterols, we questioned whether oral administration of Oxy210 in mice would result in plasma concentrations high enough to sustain significant antiviral activity in vivo. According to a previously established protocol [16], a single dose of Oxy210 at 200 mg/kg was orally administered to mice and the plasma concentration examined at 0.25, 0.5, 1, 2, 4, and 8 hours (h). Oxy210 was well tolerated by the mice in this experiment.…”

“…We have previously characterized Oxy210 as a Hh and TGFβ signaling inhibitor [16] and have demonstrated protective effects of Oxy210 in a mouse model of idiopathic pulmonary fibrosis (IPF) (Parhami et al, unpublished observations). Oxy232, a close structural analogue of Oxy210, but devoid of significant TGFβ pathway inhibitory properties, displayed anti-SARS-CoV-2 activity comparable to Oxy210, suggesting that the mechanisms of the anti-SARS-CoV-2 activity shared by Oxy210 and Oxy232 are likely unrelated to TGFβ inhibitory properties exhibited by Oxy210.…”

“…In this study, we focused on oxysterols, including naturally occurring and semi-synthetic oxysterols, to identify potent anti-SARS-CoV-2 agents, since we have already developed various semi-synthetic oxysterols as drug candidates in the context of cancer, fibrotic diseases and bone regeneration: Oxy133, an allosteric activator of Hedgehog (Hh) signaling, was designed for orthopedic applications, such as bone repair and spine fusion [12–14]; Oxy186, an inhibitor of Hh signaling that acts downstream of the Smoothened (Smo) receptor, was designed as a potential anti-tumorigenic agent [15], and Oxy210 was designed for application in cancer and fibrosis through dual inhibition of Hh and transforming growth factor-beta (TGF-β) signaling [16]. In the present report, using cell-based analysis, we demonstrate that Oxy210 and its analogue, Oxy232, display superior anti-SARS-CoV-2 activity compared to the natural oxysterols, 7-ketocholesterol (7-KC), 22( R )-hydroxycholesterol [22( R )-OHC], 24( S )-hydroxycholesterol [24( S )-OHC], and 27-OHC.…”