Bud development, flower phenology and life history of holoparasitic Rafflesia cantleyi.

Despite being the world's largest single-flower, Rafflesia's biology and life history are still poorly understood due to its cryptic growth strategy on Tetrastigma vines. Previous studies have been mostly short-term, contrary to Rafflesia's long development period before blooming. Bud development and flower phenology of R. cantleyi was studied in a dipterocarp forest in Lata Jarum, Peninsular Malaysia. Seven populations, consisting of 247 buds, were monitored fortnightly for 65 months in two discrete studies between 2009 and 2018. The bud size distribution of R. cantleyi is dynamic, progressively changing from small flower buds to larger buds before flowering. Buds < 5.0 cm across had the slowest growth and highest mortality rates, while those > 15.0 cm across demonstrated accelerated growth. The bud growth profiles of the same site clustered distinctively regardless of sex with successful blooming rate that varied greatly between sites, prompting speculation about their relatedness to the sites' physical attributes. We reported the first female-dominated population in Rafflesia's life history. Rafflesia cantleyi's blooming rate at Lata Jarum is moderate to high, with non-seasonal flowering phenology as evident by the lack of synchronisation and consistency between flowering and local rainfall patterns. Based on the field data of the present study and the published information of other Rafflesia species, R. cantleyi's life cycle was estimated between 4.0 and 5.3 years. Our findings further explain Rafflesia's biology and life history and highlight the gap in knowledge of the natural habitats on the endoparasite's growth and fate potentially for future conservation and study.

Transcriptional analysis highlights three distinct immune profiles of high-risk oral epithelial dysplasia.

Oral potentially malignant disorders (OPMD) are precursors of oral squamous cell carcinoma (OSCC), and the presence of oral epithelial dysplasia (OED) in OPMD confers an increased risk of malignant transformation. Emerging evidence has indicated a role for the immune system in OPMD disease progression; however, the underlying immune mechanisms remain elusive. In this study, we used immune signatures established from cancer to delineate the immune profiles of moderate and severe OED, which are considered high-risk OPMD. We demonstrated that moderate and severe OEDs exhibit high lymphocyte infiltration and upregulation of genes involved in both immune surveillance (major histocompatibility complex-I, T cells, B cells and cytolytic activity) and immune suppression (immune checkpoints, T regulatory cells, and tumor-associated macrophages). Notably, we identified three distinct subtypes of moderate and severe OED: immune cytotoxic, non-cytotoxic and non-immune reactive. Active immune surveillance is present in the immune cytotoxic subtype, whereas the non-cytotoxic subtype lacks CD8 immune cytotoxic response. The non-immune reactive subtype showed upregulation of genes involved in the stromal microenvironment and cell cycle. The lack of T cell infiltration and activation in the non-immune reactive subtype is due to the dysregulation of CTNNB1, PTEN and JAK2. This work suggests that moderate and severe OED that harbor the non-cytotoxic or non-immune reactive subtype are likely to progress to cancer. Overall, we showed that distinct immune responses are present in high-risk OPMD, and revealed targetable pathways that could lead to potential new approaches for non-surgical management of OED.

Effects of palbociclib on oral squamous cell carcinoma and the role of PIK3CA in conferring resistance.

OBJECTIVE: Lack of effective therapies remains a problem in the treatment of oral squamous cell carcinoma (OSCC), especially in patients with advanced tumors. OSCC development is driven by multiple aberrancies within the cell cycle pathway, including amplification of cyclin D1 and loss of p16. Hence, cell cycle inhibitors of the CDK4/6-cyclin D axis are appealing targets for OSCC treatment. Here, we determined the potency of palbociclib and identified genetic features that are associated with the response of palbociclib in OSCC. METHODS: The effect of palbociclib was evaluated in a panel of well-characterized OSCC cell lines by cell proliferation assays and further confirmed by in vivo evaluation in xenograft models. PIK3CA-mutant isogenic cell lines were used to investigate the effect of PIK3CA mutation towards palbociclib response. RESULTS: We demonstrated that 80% of OSCC cell lines are sensitive to palbociclib at sub-micromolar concentrations. Consistently, palbociclib was effective in controlling tumor growth in mice. We identified that palbociclib-resistant cells harbored mutations in PIK3CA. Using isogenic cell lines, we showed that PIK3CA mutant cells are less responsive to palbociclib as compared to wild-type cells with concurrent upregulation of CDK2 and cyclin E1 protein levels. We further demonstrated that the combination of a PI3K/mTOR inhibitor (PF-04691502) and palbociclib completely controlled tumor growth in mice. CONCLUSIONS: This study demonstrated the potency of palbociclib in OSCC models and provides a rationale for the inclusion of PIK3CA testing in the clinical evaluation of CDK4/6 inhibitors and suggests combination approaches for further clinical studies.

IFITM3 knockdown reduces the expression of CCND1 and CDK4 and suppresses the growth of oral squamous cell carcinoma cells.

PURPOSE: Oral squamous cell carcinoma (OSCC) is a challenging disease to treat. Up to 50% of OSCC patients with advanced disease develop recurrences. Elucidation of key molecular mechanisms underlying OSCC development may provide opportunities to target specific genes and, thus, to improve patient survival. In this study, we examined the expression and functional role of interferon transmembrane protein 3 (IFITM3) in OSCC development. METHODS: The expression of IFITM3 in OSCC and normal oral mucosal tissues was assessed by qRT-PCR and immunohistochemistry. The role of IFITM3 in driving OSCC cell proliferation and survival was examined using siRNA-mediated gene knockdown, and the role of IFITM3 in driving cell cycle regulators was examined using Western blotting. RESULTS: We found that IFITM3 is overexpressed in more than 79% of primary OSCCs. We also found that IFITM3 knockdown led to impaired OSCC cell growth through inhibition of cell proliferation, induction of cell cycle arrest, senescence and apoptosis. In addition, we found that IFITM3 knockdown led to reduced expressions of CCND1 and CDK4 and reduced RB phosphorylation, leading to inhibition of OSCC cell growth. This information may be instrumental for the design of novel targeted therapeutic strategies. CONCLUSIONS: From our data we conclude that IFITM3 is overexpressed in OSCC and may regulate the CCND1-CDK4/6-pRB axis to mediate OSCC cell growth.

The 4717C > G polymorphism in periplakin modulates sensitivity to EGFR inhibitors.

The use of EGFR inhibitors on oral squamous cell carcinoma (OSCC) as monotherapy yielded modest clinical outcomes and therefore would benefit from biomarkers that could predict which patient subsets are likely to respond. Here, we determined the efficacy of erlotinib in OSCC cell lines, and by comparing sensitive and resistant lines to identify potential biomarkers. We focused on the 4717C > G polymorphism in periplakin (PPL) where the CC genotype was associated with erlotinib resistance. To validate this, erlotinib-resistant cell lines harbouring CC genotype were engineered to overexpress the GG genotype and vice versa. Isogenic cell lines were then studied for their response to erlotinib treatment. We demonstrated that overexpression of the GG genotype in erlotinib-resistant lines sensitized them to erlotinib and inhibition of AKT phosphorylation. Similarly, the expression of the CC genotype conferred resistance to erlotinib with a concomitant increase in AKT phosphorylation. We also demonstrated that cell lines with the CC genotype generally are more resistant to other EGFR inhibitors than those with the GG genotype. Overall, we showed that a specific polymorphism in the PPL gene could confer resistance to erlotinib and other EGFR inhibitors and further work to evaluate these as biomarkers of response is warranted.

DeSigN: connecting gene expression with therapeutics for drug repurposing and development.

BACKGROUND: The drug discovery and development pipeline is a long and arduous process that inevitably hampers rapid drug development. Therefore, strategies to improve the efficiency of drug development are urgently needed to enable effective drugs to enter the clinic. Precision medicine has demonstrated that genetic features of cancer cells can be used for predicting drug response, and emerging evidence suggest that gene-drug connections could be predicted more accurately by exploring the cumulative effects of many genes simultaneously. RESULTS: We developed DeSigN, a web-based tool for predicting drug efficacy against cancer cell lines using gene expression patterns. The algorithm correlates phenotype-specific gene signatures derived from differentially expressed genes with pre-defined gene expression profiles associated with drug response data (IC50) from 140 drugs. DeSigN successfully predicted the right drug sensitivity outcome in four published GEO studies. Additionally, it predicted bosutinib, a Src/Abl kinase inhibitor, as a sensitive inhibitor for oral squamous cell carcinoma (OSCC) cell lines. In vitro validation of bosutinib in OSCC cell lines demonstrated that indeed, these cell lines were sensitive to bosutinib with IC50 of 0.8-1.2 µM. As further confirmation, we demonstrated experimentally that bosutinib has anti-proliferative activity in OSCC cell lines, demonstrating that DeSigN was able to robustly predict drug that could be beneficial for tumour control. CONCLUSIONS: DeSigN is a robust method that is useful for the identification of candidate drugs using an input gene signature obtained from gene expression analysis. This user-friendly platform could be used to identify drugs with unanticipated efficacy against cancer cell lines of interest, and therefore could be used for the repurposing of drugs, thus improving the efficiency of drug development.