image of lab staff working — The Ahmad Lab

Ahmad Lab

Welcome to the Ahmad Lab!

We are a basic science and translational research laboratory aiming to discover the knowledge required to make advancements in the prevention and treatment of cancer as well as other hyperproliferative skin diseases.

We are working on this by delving into two primary research areas:

Mechanisms of Cancer Development and Progression:
We focus on understanding how cancer develops and evolves, specifically by investigating critical molecular and biochemical events like the cell cycle and gene/protein modification and regulation.
Prevention, Interception, and Experimental Therapeutics of Cancer:
Our exploration extends to finding ways to prevent, intercept, and treat cancer using naturally occurring non-toxic agents as well as small molecule chemical inhibitors of key cancer pathways.

Mechanisms of Cancer Development and Progression.

One of the main goals of the Ahmad Lab revolves around investigating essential molecular and biochemical events involved in cancer development. Our specific emphasis lies in the identification and functional characterization of new targets for cancer management, including 1) the polo-like kinases and 2) sirtuins, among others. Additionally, we are dedicated to discovering crucial biomarkers for both diagnosing cancer and predicting its prognosis. Although we have a considerable breadth of projects to draw on, some of our recent projects and/or important studies are outlined here:

1. Exploring the crucial role of polo-like kinases (PLKs) in cancer.

Diagram of DNA damage control by the American Association for Cancer Research 2013. — **PLK1-interacting pathways.** PLK1 overexpression and the resulting contribution to aberrant DNA damage control and genomic instability are accentuated by multiple feedback loops highlighting the interaction of PLK1 with several pathways involved in cell-cycle progression and the DNA damage response (A); and eight of 12 signaling pathways (middle ring) and all three core cellular processes (outer ring) to confer a selective growth advantage in cancer (B). PLK1 has a direct interaction with a wide range of proteins (inner ring) that are transcribed by oncogenic (green) or tumor-suppressive (red) driver genes. Furthermore, PLK1 directly interacts with several proteins that are integral to the described pathways. From Cancer Research, 2013, 73(23):6848-55 (PMID: 24265276).

Polo-like kinases (PLKs) are a family of serine/threonine kinases that play a pivotal role as regulators in the mitotic progression in mammalian cells. Our research is focused on comprehending the functional significance of PLKs in the initiation and/or advancement of various cancers, particularly in skin and prostate cancers. Studies conducted in our laboratory have demonstrated the proliferative function of PLK1 in melanoma (Journal of Investigative Dermatology, 2009, 129(12):2843-53, PMID: 19554017; Cancer letters, 2017, 385:179-187, PMID: 27793694) and prostate cancer (FASEB Journal, 2005, 19(6):611-3, PMID: 15661849). These findings suggest that inhibiting PLK1 could serve as a rational approach for managing these specific types of cancers. Moreover, our research has unveiled that targeted knockdown of PLK1 leads to alteration in metabolic regulation in melanoma (Cancer Letters, 2017, 394:13-21, PMID: 28235541). We have also contributed to the larger body of PLK1-related knowledge by publishing a comprehensive review on the role of PLK1 in carcinogenesis (Cancer Research, 2013, 73(23):6848-55, PMID: 24265276), further illuminating the significance of PLKs in the development and progression of cancer. Please see the figure from our publication.

Graph of PLK1, NUMB, and NOTCH signaling in melanoma progression. — **PLK1, NUMB, and NOTCH signaling in melanoma progression.** Based on our in vitro human melanoma cell models and ex vivo human melanoma tissue microarray (TMA) study as well as TCGA melanoma database analysis, we propose a novel PLK1 phosphorylation site on NUMB protein that might be involved in melanoma cell migration, invasion, and EMT. Additionally, PLK1 inhibition decreases the NOTCH pathway and EMT-related molecular consequences, which may ultimately lead to improved survival of patients with melanoma. From npjPrecision Oncology, 2024, (In Press).

Our laboratory is also actively engaged in exploring the downstream mechanisms and interactions of PLK1 in cancer. In our research, we uncovered a significant interaction between PLK1 and NUMB, an antagonist of NOTCH signaling. Our findings showcased that NUMB plays a pivotal role in regulating the stability and localization of PLK1 and is required for transit through mitosis. This study unraveled a previously unexplored function of NUMB during symmetric cell division, and a potential contributing mechanism in tumors with reduced NUMB expression (Cancer Research, 2012, 72(15):3864-72, PMID: 22593191). In a recent study, we identified a novel PLK1 phosphorylation site on NUMB and a potential role of PLK1, NUMB, NOTCH signaling in epithelial-mesenchymal transition (EMT) as well as migration and invasion of melanoma cells. Our findings support the therapeutic targeting of PLK1, NUMB, and NOTCH in the clinical management of melanoma (Nature Precision Oncology, 2024, 8(1):6. doi: 10.1038/s41698-023-00493-7, PMID: 38184733). Please see the figure from our publication.

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Graphical abstract representing PLK1 inhibition-based combination therapies for cancer management. — **PLK1 inhibition-based combination therapies for cancer management.** PLK1 has been shown to have oncogenic function in several cancers. Although PLK1 inhibitors, such as BI2536, BI6727 (volasertib) and NMS-1286937 (onvansertib) are generally well-tolerated with a favorable pharmacokinetic profile, clinical successes are limited due to partial responses in cancer patients, especially those in advanced stages. Recently, combination therapies targeting multiple pathways are being tested for cancer management. PLK1-based combination therapies have shown increased apoptosis, disrupted cell cycle, and potential to overcome resistance in cancer cells/tissues over monotherapies. Further, with successes in preclinical experiments, researchers are validating such approaches in clinical trials. Understanding the mechanisms of synergism between PLK1 inhibitors and other agents may lead to a better approach on which agents to pair with PLK1 inhibition for optimum cancer treatment. From Translational Oncology, 2022,16:101332 (PMID: 34973570).

We are actively investigating innovative combination therapies for cancer management, particularly in conjunction with PLK inhibitors. In a recent review (Translational Oncology, 2022,16:101332, PMID: 34973570; see image), we discuss the rationale behind using combination therapies instead of traditional mono-therapeutic treatment regimens. Our review critically examines PLK1-based combination therapies with chemotherapy, targeted small molecule inhibitors, and immunotherapy in cancer treatments, emphasizing recent advancements and challenges. For example, one of our recently published studies demonstrates a positive correlation between PLK1 and NOTCH in melanoma, and their combined inhibition leads to synergistic modulations of key melanoma pathways (Molecular Cancer Therapeutics, 2021, 20(1):161-172, PMID: 33177155), providing compelling evidence for their combined inhibition as a potential strategy for cancer management.

Images of Biological specimen of prostate cancer and graphs depicting how PLK4 is overexpressed in human prostate cancer and its overexpression correlates with worse survival. — **PLK4 is overexpressed in human prostate cancer and its overexpression correlates with worse survival.** (A) Tissue microarray (TMA) #PR956 (US Biomax) containing prostate cancer and benign prostate specimen was immunostained using PLK4 antibody and hematoxylin as a nuclear counterstain. The representative micrographs (at ×20 magnification) showing PLK4 immunostaining are presented. Dotted areas were zoomed‐in using Nuance software. (B) The PLK4‐stained slide was scanned using Vectra system and analyzed using the inForm software for PLK4 optical density (OD) as a measure of the stain in each tissue core. (C) PLK4 level was also plotted as per Gleason grade in prostate cancer. Next, (D) disease‐free survival (DFS) and (E) overall survival (OS) were analyzed based on PLK4 mRNA expression from the 497 prostate cancer patients with available survival data from TCGA; cBioPortal was used to query the data. PLK4 mRNA Z score of 0 was used as a cutoff for “low” versus “high” PLK4 expression. (F) PLK4 mRNA expression levels based on Gleason score. From Prostate, 2022, 82(9):957-969 (PMID: 35333404).

In addition to our work on PLK1, we are actively researching the role of other PLK family members in cancer, including PLK4. Not long ago, in a joint study with another lab at UW, we found that centriole overduplication is the predominant mechanism leading to centrosome amplification in melanoma and that we believe PLK4 should be further evaluated as a potential therapeutic target for melanoma treatment. (Molecular Cancer Research, 2018, 16(3):517-527, PMID: 29330283). Other ongoing projects explore PLK4’s functional significance in melanoma and non-melanoma skin cancer development, with the aim to develop novel strategies for the management of skin cancers.

In another recently completed collaborative study (Prostate, 2022; 82(9):957-969. PMID: 35333404), we demonstrated that PLK4 is upregulated in human prostate cancer (please see figure), and its inhibition reduces centrosome amplification and causes senescence. Specifically, our data demonstrated reduced cellular growth, viability, and colony formation of human prostate cancer cells upon treatment with PLK4 inhibitors (CFI‐400945 and centrinone‐B). Further, PLK4 inhibition was found to induce cell cycle arrest in G2/M phase and senescence‐like phenotypes in prostate cancer cells.

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Graph of Sirtuin localization and function. — **Sirtuin localization and function.** Each sirtuin is biologically unique based on their cellular location and function. With controversial roles in cancer, sirtuins possess important biological functions that are necessary for cellular homeostasis. From Experimental Dermatology 2020, 29(2):124-135, (PMID: 31696978).

2. Role and functional significance of sirtuins (SIRTs) in cancer.

Sirtuins (SIRTs 1–7) are a family of nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylases with the ability to deacetylate histone and nonhistone targets. Sirtuins have been shown to undergo nuclear-cytoplasmic shuttling, where their functions within the cell appear to change depending on each tissue and cell type. Sirtuins have been shown to play a dual role in cancer as either a tumor promoter or oncogene by targeting different cellular pathways. Despite the structural similarities, each sirtuin has its own role in modulating essential cellular mechanisms to maintain homeostasis. Our recent review article (Experimental Dermatology, 2020, 29(2):124-135, PMID: 31696978) summarizes their biological roles, cellular localization, and effects on tumorigenesis (please see figure).

images of biological specimens and graph depicting the Effect of melatonin supplementation in transgenic adenocarcinoma of mouse prostate. — **Effect of melatonin supplementation in transgenic adenocarcinoma of mouse prostate (TRAMP) model.** Sixteen-week old TRAMP mice were supplemented with melatonin (0, 10 and 20 mg/L in water) for 18 weeks. A) Effect of melatonin treatment on urogenital tract (UGT) and prostate size: At the termination of experiment (at 34 weeks of age), UGT apparatus and prostates were removed and representative pictures from each group are shown; B) Effect of melatonin on prostate weight: Following dissection, prostates from each mouse were weighed and recorded. The data were obtained from each group (n=12) and are expressed as mean ± SE (*p ≤ 0.01). From Journal of Pineal Research, 2011, 50(2):140-9 (PMID: 21062352).

We are actively exploring the role of sirtuins in the development and progression of cancer, with specific emphasis on skin and prostate cancers. Our research has highlighted the potential role of SIRT1 in prostate cancer (Journal of Biological Chemistry, 2009, 284(6):3823-32, PMID: 19075016). Additionally, our findings have shown that the pineal hormone melatonin is a novel SIRT1 inhibitor and imparts anti-cancer effects against prostate cancer (Journal of Pineal Research, 2011, 50(2):140-9, PMID: 21062352) as well as resynchronizes dysregulated circadian rhythm circuitry in human prostate cancer cells (Journal of Pineal Research, 2010, 49(1):60-8, PMID: 20524973). Please see the figure from our publication.

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Graph of heat map and volcano plot of 770 cancer immune genes affected by 4'-BR treatment. — **4′-BR significantly modulated the genes associated with cancer-immune signaling, as determined using NanoString PanCancer Immune Profiling Panel.** RNA from primary tumor tissues of control and 4′-BR‒treated groups were subjected to NanoString analysis of 770 immune-related genes. (a) Left: heatmap displaying each gene with color coding for upregulated or downregulation (orange and blue, respectively). Right: volcano plots displaying each gene’s ‒log10(P-value) and log2(fold change) in 4′-BR‒treated group normalized with that in the control group. Some of the most statistically significant genes are labeled in the plot. (b) Cancer-immune pathways significantly (∗P < 0.05) modulated by 4′-BR treatment. Data are presented as mean ± SEM (n = 6) of the pathway score. 4′-BR, 4′-bromo-resveratrol. From Journal of Investigative Dermatology, 2022, 142(4):1145-1157.e7 (PMID: 34597611).

In addition to our work in prostate cancer, we are investigating the role of sirtuins in melanoma. Our research reveals that SIRT1 is overexpressed and delocalized in melanoma cells. Inhibiting SIRT1 chemically led to decreased cell growth and viability of melanoma cells by activating the tumor suppressor protein TP53 (Archives of Biochemistry and Biophysics, 2014, 563:94-100, PMID: 24751483). Additionally, our work demonstrates the pro-proliferative function of SIRT3 in melanoma. We found that SIRT3 was overexpressed in melanoma and its suppression significantly inhibited tumorigenesis in a xenograft mouse model (Journal of Investigative Dermatology, 2016, 136(4):809-818, PMID: 26743598) and altered key metabolic regulators in melanoma (Frontiers in Oncology, 2021, 11:676077, PMID: 33937086).

We have also demonstrated that dual inhibition of SIRT1 and SIRT3 by 4′-bromo-resveratrol (4′-BR; a small-molecule dual inhibitor of SIRT1 and SIRT3), in a BrafV600E/PtenNULL mouse model significantly inhibited melanoma tumors as well as lung metastasis with no adverse effects. This is likely due to downregulation of genes related to metastasis promotion, chemokine/cytokine regulation, and innate/adaptive immune functions (Journal of Investigative Dermatology, 2022, 142(4):1145-1157.e7, PMID: 34597611). Please see a figure from this publication.

Graph of Sirtuin 6 tumor suppressor and oncogenic functions. — **Sirtuin 6 tumor suppressor and oncogenic functions.** SIRT6 can act as both a tumor promoter and oncogene in cancer by targeting different cellular pathways. From Experimental Dermatology, 2020, 29(2):124-135, (PMID: 31696978).

Similarly, we have shown a potential oncogenic role of SIRT6 in melanoma (Genes & Cancer, 2017, 8(9-10):701-712, PMID: 29234488; Photochemistry and Photobiology, 2020, 96(6):1314-1320, PMID: 32621766). In a review article (Experimental Dermatology, 2020, 29(2):124-135, PMID: 31696978), we discuss tumor suppressor and oncogenic functions of SIRT6 (Please see figure). We are conducting further studies to uncover the mechanistic basis of sirtuin dysregulation in melanoma, aiming to devise strategies for melanoma management including identifying novel combinations of immunotherapy with targeted therapy.

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Prevention, Interception, and Experimental Therapeutics of Cancer.

Another major area of research focus of the Ahmad Lab is to discover innovative agents for cancer management. Our research has extensively explored the chemopreventive potential of various agents, including resveratrol (an antioxidant found in grapes, nuts, berries, and red wine), melatonin (secretory product of the pineal gland known for its antioxidant properties), vitamin E (a key antioxidant in biomembranes) and selenium (an essential micronutrient for human health).

Over the last two decades, in vitro and in vivo research from our laboratory has shown that resveratrol possesses chemopreventive potential in skin and prostate cancers. Resveratrol (trans-3,5,4’-trihydroxystilbene), an antioxidant found in grapes, has been shown to provide chemopreventive and therapeutic effects against several cancers. We have demonstrated that a topical application of resveratrol to SKH-1 hairless mice resulted in significant inhibitions of UVB-mediated increases in (i) skin edema, (ii) inflammation, (iii) cyclooxygenase (COX) and ornithine decarboxylase (ODC), and (iv) generation of hydrogen peroxide (H2O2) and lipid peroxide formation, in the skin (Toxicology and Applied Pharmacology, 2003; 186(1):28-37. PMID: 12583990).

Our research has demonstrated that resveratrol protects against multiple UVB exposure-mediated damages to the skin of SKH-1 hairless mouse model, possibly via modulation in i) cki–cyclin–cdk network and (ii) MAPK-pathway (Oncogene, 2004; 23(30):5151-60. PMID: 15122319). Additionally, we established that topical resveratrol resulted in significant inhibition of UVB exposure-mediated increases in (i) cellular proliferation (Ki-67 immunostaining), (ii) protein levels of epidermal COX-2 and ODC, (iii) protein and mRNA levels of survivin, and (iv) phosphorylation of survivin; in the skin of SKH-1 hairless mice (Photochemistry and Photobiology, 2005; 81(1):25-31. PMID: 15469386). In another important study, we confirmed that topical application of resveratrol (both pre- and post-treatment) resulted in a significant (i) inhibition in skin tumors, and (ii) delay in the onset of tumors, in a photocarcinogenesis model of skin cancer (FASEB Journal, 2005;19(9):1193-5. PMID: 15837718).

Overall, our research suggests that resveratrol possesses promise against a wide range of cutaneous disorders including skin aging and skin cancers.
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Graph of Proposed mechanisms of the cutaneous biological response to UV damage with dietary grape. — **Proposed mechanisms of the cutaneous biological response to UV damage with dietary grape.** Summary of the mechanisms explored in our grape-mediated chemoprevention studies. We found that chemoprotective effects of dietary grape were associated with reductions in oxidative stress, survival, and proliferation. The anti-inflammatory response of dietary grape was associated with the modulation of acute-phase proteins and therefore, upstream regulators (NF-κB and MAPK). Red arrows indicate an upregulation of the protein or process. Green arrows indicate downregulation of protein or process. From Journal of Proteome Research, 2019, 18(10):3741-3751 (PMID: 31487184).

In an in vivo study, employing a mouse model of UVB-induced skin carcinogenesis, we demonstrated that consumption of 3% or 5% whole grape powder (containing resveratrol as well as several other antioxidants) in the diet significantly inhibited tumor incidence and delayed the onset of tumor growth (Journal of Investigative Dermatology, 2019; 139(3):552-561, PMID: 30393084). The addition of grape powder to the diet showed numerous effects, including enhanced DNA damage repair, decreased cell proliferation, increased cancer cell death, and favorable changes in several markers of oxidative stress.

In a subsequent study, we attempted to identify molecular targets of dietary grape-mediated chemoprevention of UVB skin carcinogenesis by conducting a comparative quantitative proteomics analysis (Journal of Proteome Research, 2019, 18(10):3741-3751, PMID: 31487184). This study demonstrated that the observed chemoprotective response of grape powder supplementation was associated with the modulation of acute-phase proteins and upstream regulators (including NF-κB and MAPK). Consuming grape powder also modulated the proteasome activity, which plays a role in cell cycle regulation and disposal of damaged proteins that contribute to cancer progression. Please see the figure from this study.

In addition, we studied the combinatorial effect of two grape antioxidants, resveratrol and quercetin, in the prevention (where supplementation was started before the onset of the disease) and intervention (supplementation started once disease was already established) settings using the TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mouse model of prostate cancer. We demonstrated that the resveratrol–quercetin combination was markedly superior to either of the individual agents, especially in the intervention setting when treatment was started after the onset of prostate cancer (Cancers, 2020; 12(8):2141, PMID: 32748838). The observed effects were associated with marked inhibition in proliferation, oxidative stress, and tumor survival markers, as well as induction of apoptosis markers.

Graph of Tissue collection and molecular analysis. — **Dietary supplementation of grape antioxidants quercetin and resveratrol inhibit tumor development and progression in Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model.** Preclinical study design and the timeline for prevention and intervention settings in TRAMP mice. From Cancers, 2020; 12(8):2141 (PMID: 32748838).

Utilizing prostate cancer PCR array analysis with prevention tumor tissues, we identified that supplementation with resveratrol and quercetin modulates genes involved in promoter methylation, cell cycle, apoptosis, fatty acid metabolism, transcription factors, androgen response, PI3K/AKT and PTEN signaling. Ingenuity Pathway Analysis (IPA) identified IGF1 and BCL2 as central players in two gene networks. Functional annotation predicted increased apoptosis and inhibited cell viability/proliferation, hyperplasia, vasculogenesis, and angiogenesis with dual treatment. Furthermore, IPA predicted upstream inhibition of major prostate cancer signaling (VEGF, Ca2+, PI3K, CSF2, PTH). Based on PCR array, we identified decreased levels of EGFR, EGR3, and IL6, and increased levels of IGFBP7 and NKX3.1, overall supporting anti- prostate cancer effects of resveratrol-quercetin. Please see the figure from our published study.

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