ASCL1-regulated DARPP-32 and t-DARPP stimulate small cell lung cancer growth and neuroendocrine tumour cell proliferation.
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ABSTRACT: Background
Small cell lung cancer (SCLC) is the most aggressive form of lung cancer, and new molecular insights are necessary for prognostic and therapeutic advances.Methods
Dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP-32) and its N-terminally truncated splice variant, t-DARPP, were stably overexpressed or ablated in human DMS-53 and H1048 SCLC cells. Functional assays and immunoblotting were used to assess how DARPP-32 isoforms regulate SCLC cell growth, proliferation, and apoptosis. DARPP-32-modulated SCLC cells were orthotopically injected into the lungs of SCID mice to evaluate how DARPP-32 and t-DARPP regulate neuroendocrine tumour growth. Immunostaining for DARPP-32 proteins was performed in SCLC patient-derived specimens. Bioinformatics analysis and subsequent transcription assays were used to determine the mechanistic basis of DARPP-32-regulated SCLC growth.Results
We demonstrate in mice that DARPP-32 and t-DARPP promote SCLC growth through increased Akt/Erk-mediated proliferation and anti-apoptotic signalling. DARPP-32 isoforms are overexpressed in SCLC patient-derived tumour tissue, but undetectable in physiologically normal lung. Achaete-scute homologue 1 (ASCL1) transcriptionally activates DARPP-32 isoforms in human SCLC cells.Conclusions
We reveal new regulatory mechanisms of SCLC oncogenesis that suggest DARPP-32 isoforms may represent a negative prognostic indicator for SCLC and serve as a potential target for the development of new therapies.
SUBMITTER: Alam SK
PROVIDER: S-EPMC7463034 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundDarpp-32 and t-Darpp are expressed in several forms of breast cancer. Both are transcribed from the gene PPP1R1B via alternative promoters. In humans, Darpp-32 is expressed in both normal and malignant breast tissue, whereas t-Darpp has only been found in malignant breast tissue. The exact biological functions of these proteins in the breast are not known. Although Darpp-32 is a well known regulator of neurotransmission, its role in other tissues and in cancer is less well understood. t-Darpp is known to increase cellular growth, inhibit apoptosis and contribute to acquired drug resistance. The use of transgenic mouse mammary tumor models to study Darpp-32 and t-Darpp in breast cancer in vivo has been limited by a lack of knowledge regarding t-Darpp expression in mice, in both normal and malignant tissue.MethodsWe used RT-PCR and Western analysis to investigate Darpp-32 and t-Darpp levels in normal and malignant mouse mammary tissue. To determine if Darpp-32 and t-Darpp play a direct role in mammary tumor development, Ppp1r1b gene knockout mice and wild-type mice were crossed with a mouse mammary tumor model. Tumor growth and metastasis were examined. Differences between groups were determined by the two-tailed Student's t-test.ResultsWe found that Darpp-32 was expressed in normal mouse mammary tissue and in some breast tumors, whereas t-Darpp was found exclusively in tumors, with t-Darpp usually expressed at equal or higher levels than Darpp-32. Ppp1r1b knockout in MMTV-PyMT transgenic tumor mice resulted in a decrease in tumor growth.ConclusionsThe shift in expression from Darpp-32 to t-Darpp during mouse mammary tumorigenesis is reminiscent of the expression patterns observed in humans and is consistent with a role for t-Darpp in promoting cell growth and Darpp-32 in inhibiting cell growth. Decreased tumor growth in Ppp1r1b knockout mice also suggests that t-Darpp plays a direct role, predominant to Darpp-32, in mammary tumor development. These results indicate that transgenic mouse mammary tumor models might be valuable tools for future investigation of Darpp-32 and t-Darpp in breast cancer.
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Project description:DARPP-32 (PPP1R1B) was discovered as a substrate of cAMP-dependent protein kinase (PKA) enriched in dopamine-innervated brain areas. It is one of three related, PKA-regulated inhibitors of protein phosphatase-1 (PP1). These inhibitors seem to have appeared in early vertebrate ancestors, possibly Gnathostomes. DARPP-32 has additional important biochemical properties including inhibition of PKA when phosphorylated by Cdk5 and regulation by casein kinases 1 and 2. It is highly enriched in specific neuronal populations, especially striatal medium-size spiny neurons. As PP1 inhibitor DARPP-32 amplifies and/or mediates many actions of PKA at the plasma membrane and in the cytoplasm, with a broad spectrum of potential targets and functions. DARPP-32 also undergoes a continuous and tightly regulated cytonuclear shuttling. This trafficking is controlled by phosphorylation of Ser-97, which is necessary for nuclear export. When phosphorylated on Thr-34 and dephosphorylated on Ser-97, DARPP-32 can inhibit PP1 in the nucleus and modulate signaling pathways involved in the regulation of chromatin response. Recent work with multiple transgenic and knockout mutant mice has allowed the dissection of DARPP-32 function in striato-nigral and striato-pallidal neurons. It is implicated in the action of therapeutic and abused psychoactive drugs, in prefrontal cortex function, and in sexual behavior. However, the contribution of DARPP-32 in human behavior remains poorly understood. Post-mortem studies in humans suggest possible alterations of DARPP-32 levels in schizophrenia and bipolar disorder. Genetic studies have revealed a polymorphism with possible association with psychological and psychopathological traits. In addition, a short isoform of DARPP-32, t-DARPP, plays a role in cancer, indicating additional signaling properties. Thus, DARPP-32 is a non-essential but tightly regulated signaling hub molecule which may improve the general performance of the neuronal circuits in which it is expressed.
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