Reciprocal antagonism of PIN1-APC/CCDH1 governs mitotic protein stability and cell cycle entry.

Induced oncoproteins degradation provides an attractive anti-cancer modality. Activation of anaphase-promoting complex (APC/CCDH1) prevents cell-cycle entry by targeting crucial mitotic proteins for degradation. Phosphorylation of its co-activator CDH1 modulates the E3 ligase activity, but little is known about its regulation after phosphorylation and how to effectively harness APC/CCDH1 activity to treat cancer. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1)-catalyzed phosphorylation-dependent cis-trans prolyl isomerization drives tumor malignancy. However, the mechanisms controlling its protein turnover remain elusive. Through proteomic screens and structural characterizations, we identify a reciprocal antagonism of PIN1-APC/CCDH1 mediated by domain-oriented phosphorylation-dependent dual interactions as a fundamental mechanism governing mitotic protein stability and cell-cycle entry. Remarkably, combined PIN1 and cyclin-dependent protein kinases (CDKs) inhibition creates a positive feedback loop of PIN1 inhibition and APC/CCDH1 activation to irreversibly degrade PIN1 and other crucial mitotic proteins, which force permanent cell-cycle exit and trigger anti-tumor immunity, translating into synergistic efficacy against triple-negative breast cancer.

Reciprocal inhibition of PIN1 and APC/CCDH1 controls timely G1/S transition and creates therapeutic vulnerability.

Cyclin-dependent kinases (CDKs) mediated phosphorylation inactivates the anaphase-promoting complex (APC/CCDH1), an E3 ubiquitin ligase that contains the co-activator CDH1, to promote G1/S transition. PIN1 is a phosphorylation-directed proline isomerase and a master cancer signaling regulator. However, little are known about APC/CCDH1 regulation after phosphorylation and about PIN1 ubiquitin ligases. Here we uncover a domain-oriented reciprocal inhibition that controls the timely G1/S transition: The non-phosphorylated APC/CCDH1 E3 ligase targets PIN1 for degradation in G1 phase, restraining G1/S transition; APC/CCDH1 itself, after phosphorylation by CDKs, is inactivated by PIN1-catalyzed isomerization, promoting G1/S transition. In cancer, PIN1 overexpression and APC/CCDH1 inactivation reinforce each other to promote uncontrolled proliferation and tumorigenesis. Importantly, combined PIN1- and CDK4/6-inhibition reactivates APC/CCDH1 resulting in PIN1 degradation and an insurmountable G1 arrest that translates into synergistic anti-tumor activity against triple-negative breast cancer in vivo. Reciprocal inhibition of PIN1 and APC/CCDH1 is a novel mechanism to control timely G1/S transition that can be harnessed for synergistic anti-cancer therapy.

PARP-inhibition reprograms macrophages toward an anti-tumor phenotype.

Poly(ADP)ribosylation inhibitors (PARPis) are toxic to cancer cells with homologous recombination (HR) deficiency but not to HR-proficient cells in the tumor microenvironment (TME), including tumor-associated macrophages (TAMs). As TAMs can promote or inhibit tumor growth, we set out to examine the effects of PARP inhibition on TAMs in BRCA1-related breast cancer (BC). The PARPi olaparib causes reprogramming of TAMs toward higher cytotoxicity and phagocytosis. A PARPi-related surge in NAD+ increases glycolysis, blunts oxidative phosphorylation, and induces reverse mitochondrial electron transport (RET) with an increase in reactive oxygen species (ROS) and transcriptional reprogramming. This reprogramming occurs in the absence or presence of PARP1 or PARP2 and is partially recapitulated by addition of NAD derivative methyl-nicotinamide (MNA). In vivo and ex vivo, the effect of olaparib on TAMs contributes to the anti-tumor efficacy of the PARPi. In vivo blockade of the "don't-eat-me signal" with CD47 antibodies in combination with olaparib improves outcomes in a BRCA1-related BC model.

Serum Response Factor (SRF) Drives the Transcriptional Upregulation of the MDM4 Oncogene in HCC.

Different molecular mechanisms support the overexpression of the mouse double minute homolog 4 (MDM4), a functional p53 inhibitor, in human hepatocellular carcinoma (HCC). However, the transcription factors (TFs) leading to its transcriptional upregulation remain unknown. Following promoter and gene expression analyses, putative TFs were investigated using gene-specific siRNAs, cDNAs, luciferase reporter assays, chromatin immunoprecipitation, and XI-011 drug treatment in vitro. Additionally, MDM4 expression was investigated in SRF-VP16iHep transgenic mice. We observed a copy-number-independent upregulation of MDM4 in human HCCs. Serum response factor (SRF), ELK1 and ELK4 were identified as TFs activating MDM4 transcription. While SRF was constitutively detected in TF complexes at the MDM4 promoter, presence of ELK1 and ELK4 was cell-type dependent. Furthermore, MDM4 was upregulated in SRF-VP16-driven murine liver tumors. The pharmacological inhibitor XI-011 exhibited anti-MDM4 activity by downregulating the TFs driving MDM4 transcription, which decreased HCC cell viability and increased apoptosis. In conclusion, SRF drives transcriptional MDM4 upregulation in HCC, acting in concert with either ELK1 or ELK4. The transcriptional regulation of MDM4 may be a promising target for precision oncology of human HCC, as XI-011 treatment exerts anti-MDM4 activity independent from the MDM4 copy number and the p53 status.

Identification of Pparγ-modulated miRNA hubs that target the fibrotic tumor microenvironment.

Liver fibrosis interferes with normal liver function and facilitates hepatocellular carcinoma (HCC) development, representing a major threat to human health. Here, we present a comprehensive perspective of microRNA (miRNA) function on targeting the fibrotic microenvironment. Starting from a murine HCC model, we identify a miRNA network composed of 8 miRNA hubs and 54 target genes. We show that let-7, miR-30, miR-29c, miR-335, and miR-338 (collectively termed antifibrotic microRNAs [AF-miRNAs]) down-regulate key structural, signaling, and remodeling components of the extracellular matrix. During fibrogenic transition, these miRNAs are transcriptionally regulated by the transcription factor Pparγ and thus we identify a role of Pparγ as regulator of a functionally related class of AF-miRNAs. The miRNA network is active in human HCC, breast, and lung carcinomas, as well as in 2 independent mouse liver fibrosis models. Therefore, we identify a miRNA:mRNA network that contributes to formation of fibrosis in tumorous and nontumorous organs of mice and humans.

Dysregulated serum response factor triggers formation of hepatocellular carcinoma.

UNLABELLED: The ubiquitously expressed transcriptional regulator serum response factor (SRF) is controlled by both Ras/MAPK (mitogen-activated protein kinase) and Rho/actin signaling pathways, which are frequently activated in hepatocellular carcinoma (HCC). We generated SRF-VP16iHep mice, which conditionally express constitutively active SRF-VP16 in hepatocytes, thereby controlling subsets of both Ras/MAPK- and Rho/actin-stimulated target genes. All SRF-VP16iHep mice develop hyperproliferative liver nodules that progresses to lethal HCC. Some murine (m)HCCs acquire Ctnnb1 mutations equivalent to those in human (h)HCC. The resulting transcript signatures mirror those of a distinct subgroup of hHCCs, with shared activation of oncofetal genes including Igf2, correlating with CpG hypomethylation at the imprinted Igf2/H19 locus. CONCLUSION: SRF-VP16iHep mHCC reveal convergent Ras/MAPK and Rho/actin signaling as a highly oncogenic driver mechanism for hepatocarcinogenesis. This suggests simultaneous inhibition of Ras/MAPK and Rho/actin signaling as a treatment strategy in hHCC therapy.

Frequency of EGFR mutations in 907 lung adenocarcioma patients of Indian ethnicity.

BACKGROUND: During the past decade, the incidence of EGFR mutation has been shown to vary across different ethnicities. It occurs at the rate of 10-15% in North Americans and Europeans, 19% in African-Americans, 20-30% in various East Asian series including Chinese, Koreans, and Japanese. Frequency of EGFR mutations in India however remains sparsely explored. METHODOLOGY/PRINCIPAL FINDINGS: We report 23% incidence of Epidermal growth factor receptor (EGFR) mutations in 907 Non small cell lung cancer (NSCLC) patients of Indian ethnicity, in contrast to 10-15% known in Caucasians and 27-62% among East Asians. In this study, EGFR mutations were found to be more common in never-smokers 29.4% as compared to smokers 15.3%. Consistent with other populations, mutation rates among adenocarcinoma-males were predominantly lower than females with 32% incidence. However unlike Caucasians, EGFR mutation rate among adenocarcinoma-never-smoker females were comparable to males suggesting lack of gender bias among never smokers likely to benefit from EGFR targeted therapy. CONCLUSIONS/SIGNIFICANCE: This study has an overall implication for establishing relevance for routine EGFR mutation diagnostics for NSCLC patients in clinics and emphasizes effectiveness for adoption of EGFR inhibitors as the first line treatment among Indian population. The intermediate frequency of EGFR mutation among Indian population compared to Caucasians and East Asians is reminiscent of an ancestral admixture of genetic influence from Middle Easterners, Central Asians, and Europeans on modern- Indian population that may confer differential susceptibility to somatic mutations in EGFR.

EGFR mutations in Indian lung cancer patients: clinical correlation and outcome to EGFR targeted therapy.

Screening for EGFR mutation is a key molecular test for management of lung cancer patients. Outcome of patients with mutation receiving EGFR tyrosine kinase inhibitor is known to be better across different ethnic populations. However, frequency of EGFR mutations and the clinical response in most other ethnic populations, including India, remains to be explored. We conducted a retrospective analysis of Indian lung cancer patients who were managed with oral tyrosine kinase inhibitors. Majority of the patients in the study had adenocarcinoma and were non-smokers. 39/111 patients tested positive for EGFR kinase domain mutations determined by Taqman based real time PCR. The overall response to oral TKI therapy was 30%. Patients with an activating mutation of EGFR had a response rate of 74%, while the response rate in patients with wild type EGFR was 5%, which was a statistically significant difference. Progression free survival of patients with EGFR mutations was 10 months compared to 2 months for EGFR mutation negative patients. Overall survival was 19 months for EGFR mutation patients and 13 months for mutation negative patients. This study emphasizes EGFR mutation as an important predictive marker for response to oral tyrosine kinase inhibitors in the Indian population.