Background & Aims Hepatocellular carcinoma is the third leading cause of cancer mortality worldwide; current chemotherapeutic interventions for this disease are largely ineffective. The ...retinoblastoma tumor suppressor (RB) is functionally inactivated at relatively high frequency in hepatocellular carcinoma and hepatoma cell lines. Here, we analyzed the ability of CDK4/6 inhibition to inhibit hepatocyte proliferation and the effect of RB status on this process. Methods Hepatoma cell lines and xenograft models harboring RB knockdown and mice harboring liver-specific Rb deletion were used to define the role of RB function in response to CDK4/6 inhibition. Results Our study shows that CDK4/6-dependent cell cycle progression in hepatoma cells was readily arrested by inhibition of CDK4/6 by PD-0332991 or p16ink4a irrespective of RB status. Interestingly, upon CDK4/6 inhibition, p107 protein stability was dramatically increased as a function of RB loss. This engagement of compensatory mechanisms was critical for cell cycle inhibition in the absence of RB, because both the E1A oncoprotein and overexpression of E2F proteins were capable of overcoming the effect of CDK4/6 inhibition. These findings were recapitulated in xenograft models. Furthermore, to determine how these findings relate to hepatocyte proliferation in vivo, mice were exposed to carbon tetrachloride to induce liver regeneration followed by treatment with PD-0332991. This treatment significantly inhibited hepatocyte proliferation. Strikingly, this facet of PD-0332991 function was retained even in RB-deficient livers. Conclusions These data show that CDK4/6 inhibition is a potent mediator of cytostasis and that RB loss can be readily compensated for in the context of both hepatoma cell lines and liver tissue.
Abstract
Background: Metastatic castration-resistant prostate cancer (mCRPC) is responsible for most of PCa deaths. Androgen deprivation therapy (ADT), taxanes and enzalutamide are the standard care ...for CRPC and mCRPC. Unfortunately, these approaches often fail to prevent tumor relapse due to at least in part, the inherently low oxygen levels within the prostate tumor environment. In addition to this, lower drug stability and fast drug clearance among ADT patients are the major therapeutic limitations in drug delivery and drug efficacy. Therefore, it is clinically warranted to develop an increased drug stability system to enhance the drug efficacy. Recent developments in drug delivery system including our earlier report, nanoparticle loaded drugs shows greater stability and better drug efficacy with sub-optimal drug concentrations in vitro. It is an essential to develop an ideal CRPC preclinical model system to test the anti-PSMA-drug conjugated hybrid nanoparticle efficacy with high drug stability with limited cellular toxicity.
Materials and Methods: CRPC lines were developed from PSMA proficient LNCaP, LAPC4, C4-2, 22Rv1, and PSMA deficient PC3-ML cells with intermittent hypoxia. CRPC cells were analyzed for proliferation, RB status, AR signaling, migration invasion and drug resistance in both in vitro and in vivo. Further, CRPC bone metastases clinical samples from Thomas Jefferson University Hospital were compared with preclinical CRPC model. Efficient anti-PSMA-drug conjugated hybrid nanoparticle therapy was established.
Results: Hypoxia resistance models show CRPC, RB loss, hormone therapy resistance in vitro and in vivo. Anti-PSMA-drug conjugated hybrid nanoparticle shows enhanced drug stability and drug efficacy in vitro and in vivo. Interestingly we observed fibrosis, immune impairment and inactive RB in bone metastatic CRPC samples correlated with our preclinical studies. Further, our clinical bone metastases samples present osteoblastic, osteolytic and osteoblastic & osteolytic nature. Anti-PSMA conjugated-drug loaded hybrid nanoparticles localized to PSMA specific cells in vitro & in vivo, causing cell cycle arrest. Drug loaded anti-PSMA conjugated hybrid nanoparticles did not cause major toxicities in mice.
Conclusion: Hypoxia resistant mCRPC models are ideal and are comparable with CRPC induced bone metastases in patient’s samples. Drug-loaded anti-PSMA conjugated hybrid nanoparticles targets PSMA specific prostate cancer cells and show enhanced drug stability and drug efficacy in CRPC preclinical model.
Citation Format: Thangavel Chellappagounder. Molecular characterization of CRPC pathogenesis and development of a novel anti-PSMA-drug conjugated hybrid nanoparticle therapy with reduced cellular toxicity abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1742.
Perturbations in the retinoblastoma pathway are over-represented in advanced prostate cancer; retinoblastoma loss promotes bypass of first-line hormone therapy. Conversely, preliminary studies ...suggested that retinoblastoma-deficient tumors may become sensitized to a subset of DNA-damaging agents. Here, the molecular and in vivo consequence of retinoblastoma status was analyzed in models of clinical relevance.
Experimental work was performed with multiple isogenic prostate cancer cell lines (hormone sensitive: LNCaP and LAPC4 cells and hormone resistant C42, 22Rv1 cells; stable knockdown of retinoblastoma using shRNA). Multiple mechanisms were interrogated including cell cycle, apoptosis, and DNA damage repair. Transcriptome analysis was performed, validated, and mechanisms discerned. Cell survival was measured using clonogenic cell survival assay and in vivo analysis was performed in nude mice with human derived tumor xenografts.
Loss of retinoblastoma enhanced the radioresponsiveness of both hormone-sensitive and castrate-resistant prostate cancer. Hypersensitivity to ionizing radiation was not mediated by cell cycle or p53. Retinoblastoma loss led to alteration in DNA damage repair and activation of the NF-κB pathway and subsequent cellular apoptosis through PLK3. In vivo xenografts of retinoblastoma-deficient tumors exhibited diminished tumor mass, lower PSA kinetics, and decreased tumor growth after treatment with ionizing radiation (P < 0.05).
Loss of retinoblastoma confers increased radiosensitivity in prostate cancer. This hypersensitization was mediated by alterations in apoptotic signaling. Combined, these not only provide insight into the molecular consequence of retinoblastoma loss, but also credential retinoblastoma status as a putative biomarker for predicting response to radiotherapy.
Sex differences in drug metabolism have been reported in numerous species, including humans. In rats and mice, sex-dependent differences in circulating growth hormone profiles are responsible for the ...differential expression of multiple sex-dependent isoforms of cytochrome P450, which is the basis for the sexual dimorphism in drug metabolism. In contrast, very little is known about sex differences in human isoforms of cytochrome P450 and their regulation by growth hormone. In this study, we have examined the effects of physiologic-like exposure doses to dexamethasone and/or pulsatile (masculine) or constant (feminine) human growth hormone on expression levels of CYP3A4, 1A2, 2D6, and 2E1 and the glucocorticoid and growth hormone receptors in hepatocyte cultures obtained from men and women donors. We report that growth hormone can regulate expression of CYP3A4, 1A2, and 2D6. The masculine-like pulsatile growth hormone profile suppresses dexamethasone-induced CYP3A4, 1A2, and 2D6, whereas the feminine-like constant profile is permissive allowing isoform expression to be equal to or greater than glucocorticoid induction alone. There are intrinsic sexual differences in hepatocytes of men and women resulting in different levels of responsiveness of CYP3A4, 1A2, and hormone receptor expression to the same sexually dimorphic growth hormone profiles. Last, although real, the sexually dimorphic effects of growth hormone on human cytochrome P450 expression are not as dramatic as those observed in rats and could easily be overlooked by the heterogeneous backgrounds of human populations.
Once reserved solely for the treatment of short stature, the now readily available recombinant GH has expanded the use of the hormone to include the treatment of cardiovascular, renal, muscular, ...skeletal, immunological, psychosocial, and metabolic abnormalities associated with GH deficiency. There are also proposals for the widespread use of the hormone to ameliorate or reverse aging. However, this extensive use of GH has revealed intrinsic sexual dimorphisms in which females are considerably less responsive to the therapeutic regimen than are males. Dynamic changes in the Janus kinase-2 (Jak2)/signal transducers and activators of transcription (Stat5B) signaling pathway as determined by transducer activation, Stat5B binding to the GH-responsive promoter of the CYP2C11 gene, and expression levels of the suppressors of cytokine signaling family (Socs2, Socs3, and Cis) were examined in male and female rat-derived primary hepatocyte cultures exposed to the masculine-like episodic GH profile. We report that the cellular actions of GH normally mediated by activation of the Jak2/Stat5B pathway are suppressed in female cells possibly due to an inherent overexpression of Cis, a member of the suppressors of cytokine signaling family that normally down-regulates the Jak2/Stat5B pathway.
There is an unmet need in the development of an effective therapy for mutant K-ras-expressing non-small-cell lung cancer (NSCLC). Although various small molecules have been evaluated, an effective ...therapy remains a dream. siRNAs have the potential to downregulate mutant K-ras both at the protein and mRNA levels. However, a safe and effective delivery of siRNAs to tumors remains a limitation to their translational application in the treatment of this highly debilitating disease. Here we developed a novel hybrid nanoparticle carrier for effective delivery of anti-mutant K-ras to NSCLC (AKSLHN). The ability of this treatment modality to regress lung tumors in mouse models was evaluated as a monotherapy or as a combination treatment with erlotinib. Further, the toxicity of this treatment modality to healthy tissues was evaluated, along with its ability to elicit immune/inflammatory reactions. The results suggest that this treatment modality is a promising prospect for the treatment of mutant K-ras-expressing NSCLC without any accompanying toxicity. However, further understanding of the cellular-level interaction between AHSLHN and erlotinib needs to be attained before this promising treatment modality can be brought to the bedside.
Because of its myriad physiologic functions, it is not surprising that the actions of growth hormone (GH) are mediated by recruiting/activating dozens of signaling molecules involved in numerous ...transduction pathways. The particular signal transduction pathway activated by the hormone is determined by the affected target cell, the sexually dimorphic secretory GH profile (masculine episodic or feminine continuous) to which the cell is exposed, and the individual's sex. In this regard, expression of female-specific CYP2C12, the most abundant cytochrome P450 in female rat liver, is solely regulated by the feminine GH profile. Sex is a modulating factor in this response in that males are considerably less responsive than females to the CYP2C12-induction effects of continuous GH. Using primary hepatocytes derived from male and female hypophysectomized rats, we have identified several factors in a transduction pathway activated by the feminine GH regime and associated with the induction of hepatic CYP2C12. Elements in the proposed pathway, in their likely order of activation, are the growth hormone receptor, extracellular signal-regulated kinases, the cAMP-response element-binding protein, and hepatocyte nuclear factors 4alpha and 6, which subsequently bind and activate the CYP2C12 promoter. Recruitment and/or activation levels of all of the component factors in the pathway were highly suppressed in male hepatocytes, possibly explaining the dramatically lower induction levels of CYP2C12 in males exposed to the same continuous GH profile as females.
Although in vivo expression levels of the male-specific hepatic isoforms of cytochrome P450 (P450) (CYP2C11, CYP2C13, CYP2A2, and CYP3A2) are determined by the episodic growth hormone profile ...secreted by male rats, these isoforms have been completely refractory to growth hormone regulation in hepatocyte culture. By using species-specific rat growth hormone, at subphysiologic in vivo concentrations administered in two daily episodic pulses, we successfully induced CYP2C11 and CYP2A2 to near normal concentrations. Whereas inductive levels of CYP2C13 were subnormal, CYP3A2 was unresponsive to all hormonal treatments, quickly declining to undetectable concentrations. In agreement with in vivo findings, we observed that induction levels of the isoforms were always greatest when the male hepatocytes were exposed to the masculine-like episodic growth hormone profile and least stimulated by the continuous feminine-like hormone profile. When administered alone, dexamethasone consistently increased isoform levels. However, when administered with growth hormone, the glucocorticoid was always antagonistic, suppressing growth hormone induction of CYP2C11, CYP2C13, and CYP2A2. Finally, the P450 isoforms were completely unresponsive to all treatments when the hepatocytes were derived from female rats, supporting earlier findings that expression levels of sexually dimorphic P450 isoforms are inherently irreversible between sexes.
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